Compositions and methods for controlling pain

ABSTRACT

The present disclosure provides compositions and methods for controlling pain. The present disclosure provides methods for identifying agents that control pain.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/865,962, filed Aug. 14, 2013, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. NS080954awarded by the National Institutes of Health. The government has certainrights in the invention.

INTRODUCTION

Primary nociceptors are the first neurons in a complex pain-processingsystem that regulates normal and pathological pain. The ability toexcite and inhibit these neurons for the purposes of research andtherapy has been limited by pharmacological and electrical stimulationconstraints; thus non-invasive, spatially localized control ofnociceptors in freely moving animals has not been possible.

Literature

Liske et al. (2013) Muscle & Nerve 47:916; Llewellyn et al. (2010)Nature Med. 16:1161; Ji et al. (2012) PLoS One 7:e32699; Wang and Zylka(2009) J. Neurosci. 29:13020; Daou et al. (2012) Soc. Neurosci. Conf.575.06/1I11; Daou et al. (2013) J. Neurosci. 33:47; Mourot et al. (2012)Nat. Methods 9:396; Kokel et al. (2013) Nat. Chem. Biol. 9:257; Mattiset al. (2012) Nat. Methods 9:159; Williams and Denison (2013) Sci.Trani. Med. 5:177ps6; Chow and Boyden (2013) Sci. Trani. Med. 5:177ps5;Towne et al. (2010) Gene Ther. 17:141; Towne et al. (2009) Mol. Pain5:52; Iyer et al. (2014) Nat. Biotech. 32:3.

SUMMARY

The present disclosure provides compositions and methods for controllingpain. The present disclosure provides methods for identifying agentsthat control pain.

Features

The present disclosure features a method for controlling pain in anindividual, the method comprising introducing into a nociceptor of theindividual a nucleic acid comprising a nucleotide sequence encoding anopsin polypeptide that provides for hyperpolarization of the nociceptorin response to light of a wavelength that activates the opsin. In somecases, the light is delivered transdermally. In some cases, the opsincomprises an amino acid sequence having at least about 75% amino acidsequence identity to one of SEQ ID NOs:1, 3, 4, 6, 15, and 16. In somecases, the pain is neuropathic pain. In some cases, the nucleic acidcomprising a nucleotide encoding the opsin is administered to theindividual via injection into a nerve, via intramuscular injection, orvia intravenous injection. In some cases, the nucleic acid isadministered to the individual at or near a treatment site (e.g., a siteof pain). In some cases, the nucleic acid is a recombinant expressionvector, e.g., the recombinant expression vector is a viral vector. Insome instances, where the recombinant expression vector is a viralvector, the viral vector is a lentivirus vector or an adeno-associatedvirus (AAV) vector. In some instances, the AAV vector is an AAV6 vectoror an AAV8 vector. The nucleotide sequence can be operably linked to apromoter that provides for selective expression in a neuron. Forexample, the promoter can be a synapsin-I promoter, a human synuclein 1promoter, a human Thy1 promoter, or a calcium/calmodulin-dependentkinase II alpha (CAMKIIα) promoter. The individual can be a mammal;e.g., a human, a rat, or a mouse. In some case, activation of the opsinprovides for an at least 10%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, orat least 90%, reduction in pain. In some case, activation of the opsinprovides a 100% reduction in pain, i.e., the individual experiencessubstantially no pain.

The present disclosure features a non-human animal model of neuropathicpain, where the non-human animal expresses in a nociceptor of the animala nucleic acid comprising a nucleotide sequence encoding an opsinpolypeptide that provides for depolarization of the nociceptor inresponse to light of a wavelength that activates the opsin. In somecases, the opsin comprises an amino acid sequence having at least about75% amino acid sequence identity to one of SEQ ID NOs:8-14 and 19-21. Insome cases, the nucleic acid is a recombinant expression vector, e.g., aviral vector. For example, in some cases, the viral vector is alentivirus vector or an adeno-associated virus (AAV) vector, e.g., anAAV6 vector or an AAV8 vector. In some instances, the nucleotidesequence encoding the opsin is operably linked to a promoter thatprovides for selective expression in a neuron; e.g., the promoter can bea synapsin-I promoter, a human synuclein 1 promoter, a human Thy1promoter, or a calcium/calmodulin-dependent kinase I1 alpha (CAMKI1α)promoter. In some instances, the animal is a rat. In some instances, theanimal is a mouse.

The present disclosure features a method of identifying an agent thatreduces pain, the method comprising: a) administering a test agent to asubject non-human animal; and b) determining the effect, if any, of thetest agent on pain when the depolarizing light-activated polypeptide isactivated with light, where a test agent that reduces pain in thenon-human animal, compared to the level of pain induced by lightactivation of the depolarizing light-activated polypeptide in theabsence of the test agent, indicates that the test agent is a candidateagent for reducing pain.

The present disclosure features a method of identifying an agent thatreduces pain, the method comprising: a) administering a test agent to asubject non-human animal; and b) determining the effect, if any, of thetest agent on the amount of light required to induce pain through theactivation of a depolarizing light-activated polypeptide followingadministration of the test agent, wherein a test agent that increasesthe amount of light required to produce a sign of pain, compared withthe amount of light required to produce a sign of pain in the absence ofthe test agent indicates that the test agent is a candidate agent forreducing pain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict data showing that intra-sciatic injection ofrAAV2/6-hSyn-ChR2(H134R)-eYFP transduced unmyelinated nociceptors thatprojected to spinal cord lamina.

FIGS. 2A-F depict data showing that transdermal illumination of ChR2⁺mice resulted in tunable pain-like behavior.

FIGS. 3A-G depict responses in blue light-sensitized ChR2-expressingmice and yellow light-desensitized NpHR-expressing mice to mechanicaland thermal stimuli.

FIGS. 4A and 4B depict data showing that yellow light stimulation ofNpHR′ mice reversed mechanical allodynia and thermal hyperalgesia causedby a chronic constriction injury.

FIGS. 5A-G depict amino acid sequences of various light-activatedpolypeptides.

FIGS. 6A and 6B depict size distribution of opsin transduction.

FIGS. 7A-D depict representative images of NpHR transduction observedafter intra-sciatic injection of AAV2/6-hSyn-eNpHR3.0-eYFP.

FIGS. 8A-F depict electrophysiological recording from ChR2+ and NpHR+DRG neurons.

FIG. 9 provides Table 1.

FIGS. 10A and 10B depict AAV8 transduction in the lumbar and thoracicspinal cord.

FIG. 11 depicts expression of eNpHR3.0 in trigeminal ganglion sensoryneurons following direct injection into ganglion.

FIG. 12 depicts mechanical thresholds for mice expressing GFP oreNpHR3.0 in nociceptive fibers using AAV6.

DEFINITIONS

As used herein, an “individual,” “subject,” or “patient” is an animal,e.g., a mammal, including a human. Mammals include, but are not limitedto, ungulates, canines, felines, bovines, ovines, non-human primates,lagomorphs, and rodents (e.g., mice and rats). In one aspect, anindividual is a human. In another aspect, an individual is a non-humanmammal.

Amino acid substitutions in a native protein sequence may be“conservative” or “non-conservative” and such substituted amino acidresidues may or may not be one encoded by the genetic code. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a chemicallysimilar side chain (i.e., replacing an amino acid possessing a basicside chain with another amino acid with a basic side chain). A“non-conservative amino acid substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a chemicallydifferent side chain (i.e., replacing an amino acid having a basic sidechain with an amino acid having an aromatic side chain). The standardtwenty amino acid “alphabet” is divided into chemical families based onchemical properties of their side chains. These families include aminoacids with basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) and sidechains having aromatic groups (e.g., tyrosine, phenylalanine,tryptophan, histidine).

As used herein, an “effective dosage” or “effective amount” of arecombinant expression vector, or a pharmaceutical compositioncomprising a recombinant expression vector, is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. An effectivedosage can be administered in one or more administrations. For purposesof this disclosure, an effective dosage of a recombinant expressionvector, or a pharmaceutical composition comprising a recombinantexpression vector, is an amount sufficient to accomplish prophylactic ortherapeutic treatment either directly or indirectly. For example, aneffective dosage of a recombinant expression vector, or a pharmaceuticalcomposition comprising a recombinant expression vector, can be an amountsufficient to reduce pain (e.g., neuropathic pain). As is understood inthe clinical context, an effective dosage of a recombinant expressionvector, or a pharmaceutical composition comprising a recombinantexpression vector, may or may not be achieved in conjunction withanother drug, compound, or pharmaceutical composition. Thus, an“effective dosage” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this disclosure, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, delaying the progression of the disease,and/or prolonging survival of individuals. For example, “treatment” or“treating” can refer to reduction in pain.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anopsin” includes a plurality of such opsin and reference to “thenociceptor” includes reference to one or more nociceptors andequivalents thereof known to those skilled in the art, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for controllingpain in an individual. The present disclosure provides methods foridentifying agents that control pain.

Methods of Reducing Pain

The present disclosure provides compositions and methods for controllingpain in an individual. In some cases, methods for controlling painaccording to the present disclosure generally involve introducing into aneuron of an individual a nucleic acid comprising a nucleotide sequenceencoding an opsin that provides for hyperpolarization of the cell inresponse to light of a wavelength that activates the opsin; such amethod provides for reduction of pain. The nucleic acid enters theneuron (e.g., a primary afferent neuron, such as a small- or alarge-diameter primary afferent neuron; e.g., a nociceptor), the opsinis produced in the neuron, and the opsin is inserted into the cellmembrane. The terms “opsin,” “light-responsive protein,”“light-responsive polypeptide,” “light-activated protein,” and“light-activated polypeptide,” are used interchangeably herein.

In some cases, a nucleic acid comprising a nucleotide sequence encodingan opsin that provides for hyperpolarization of the cell in response tolight of a wavelength that activates the opsin provides for expressionof the opsin in a neuron (e.g., primary afferent neuron; e.g., anociceptor). In some cases, a nucleic acid comprising a nucleotidesequence encoding an opsin that provides for hyperpolarization of thecell in response to light of a wavelength that activates the opsinprovides for expression of the opsin in a sub-population of nociceptors.Targeting expression of a light-activated polypeptide to asub-population of nociceptors can be achieved by one or more of:selection of the vector (e.g., AAV6; AAV1; AAV8; etc.); selection of apromoter; and delivery means. For example, injection into the sciaticnerve can provide for production of a light-activated polypeptide inunmyelinated nociceptors (putative C-fibers).

The present disclosure provides methods for reducing pain, e.g., painsuch as acute pain, chronic pain, neuropathic pain, nociceptive pain,allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathies,neuralgia, diabetic neuropathy, human immunodeficiency virus-relatedneuropathy, nerve injury, rheumatoid arthritic pain, osteoarthriticpain, burns, back pain, eye pain, visceral pain, cancer pain (e.g. bonecancer pain), dental pain, headache, migraine, carpal tunnel syndrome,fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain,post herpetic neuralgia, post-operative pain, post stroke pain, andmenstrual pain.

Pain can be classified as acute or chronic. Acute pain begins suddenlyand is short-lived (usually in twelve weeks or less). It is usuallyassociated with a specific cause such as a specific injury and is oftensharp and severe. It is the kind of pain that can occur after specificinjuries resulting from surgery, dental work, a strain, or a sprain.Acute pain does not generally result in any persistent psychologicalresponse. In contrast, chronic pain is long-term pain, typicallypersisting for more than three months and leading to significantpsychological and emotional problems. Common examples of chronic painare neuropathic pain (e.g. painful diabetic neuropathy, postherpeticneuralgia), carpal tunnel syndrome, back pain, headache, cancer pain,arthritic pain and chronic post-surgical pain. In some cases, a methodof the present disclosure is effective in reducing acute pain. In somecases, a method of the present disclosure is effective in reducingchronic pain.

Clinical pain is present when discomfort and abnormal sensitivityfeature among the patient's symptoms. Individuals can present withvarious pain symptoms. Such symptoms include: 1) spontaneous pain whichmay be dull, burning, or stabbing; 2) exaggerated pain responses tonoxious stimuli (hyperalgesia); and 3) pain produced by normallyinnocuous stimuli (allodynia—Meyer et al., 1994, Textbook of Pain,13-44). Although patients suffering from various forms of acute andchronic pain may have similar symptoms, the underlying mechanisms may bedifferent and may, therefore, require different treatment strategies.Pain can also therefore be divided into a number of different subtypesaccording to differing pathophysiology, including nociceptive pain,inflammatory pain, and neuropathic pain. In some cases, a method of thepresent disclosure is effective in reducing nociceptive pain. In somecases, a method of the present disclosure is effective in reducinginflammatory pain. In some cases, a method of the present disclosure iseffective in reducing neuropathic pain.

Nociceptive pain is induced by tissue injury or by intense stimuli withthe potential to cause injury. Moderate to severe acute nociceptive painis a prominent feature of pain from central nervous system trauma,strains/sprains, burns, myocardial infarction and acute pancreatitis,post-operative pain (pain following any type of surgical procedure),posttraumatic pain, renal colic, cancer pain and back pain. Cancer painmay be chronic pain such as tumor related pain (e.g. bone pain,headache, facial pain or visceral pain) or pain associated with cancertherapy (e.g. postchemotherapy syndrome, chronic postsurgical painsyndrome or post radiation syndrome). Cancer pain may also occur inresponse to chemotherapy, immunotherapy, hormonal therapy orradiotherapy. Back pain may be due to herniated or rupturedintervertebral discs or abnormalities of the lumber facet joints,sacroiliac joints, paraspinal muscles or the posterior longitudinalligament. Back pain may resolve naturally but in some patients, where itlasts over 12 weeks, it becomes a chronic condition which can beparticularly debilitating.

Neuropathic pain can be defined as pain initiated or caused by a primarylesion or dysfunction in the nervous system. Etiologies of neuropathicpain include, e.g., peripheral neuropathy, diabetic neuropathy, postherpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy,HIV neuropathy, phantom limb pain, carpal tunnel syndrome, centralpost-stroke pain and pain associated with chronic alcoholism,hypothyroidism, uremia, multiple sclerosis, spinal cord injury,Parkinson's disease, epilepsy, and vitamin deficiency.

The inflammatory process is a complex series of biochemical and cellularevents, activated in response to tissue injury or the presence offoreign substances, which results in swelling and pain. Arthritic painis a common inflammatory pain.

Other types of pain include: pain resulting from musculo-skeletaldisorders, including myalgia, fibromyalgia, spondylitis, sero-negative(non-rheumatoid) arthropathies, non-articular rheumatism,dystrophinopathy, glycogenolysis, polymyositis and pyomyositis; heartand vascular pain, including pain caused by angina, myocardicalinfarction, mitral stenosis, pericarditis, Raynaud's phenomenon,scleredoma and skeletal muscle ischemia; head pain, such as migraine(including migraine with aura and migraine without aura), clusterheadache, tension-type headache mixed headache and headache associatedwith vascular disorders; and orofacial pain, including dental pain, oticpain, burning mouth syndrome, and temporomandibular myofascial pain.

In some cases, a subject method of reducing pain involves introducinginto a nociceptor (a sensory neuron that responds to potentiallydamaging stimuli by sending nerve signals to the spinal cord and brain)a nucleic acid comprising a nucleotide sequence encoding an opsin thatprovides for hyperpolarization of the nociceptor in response to light ofa wavelength that activates the opsin, thereby reducing pain. Thenociceptor can be a thermal nociceptor, a mechanical nociceptor, achemical nociceptor, or other type of nociceptor. Nociceptive markersinclude, but are not limited to, IB4, Substance P, TRPV1, andsomatostatin.

Whether pain is reduced can be determined in a human subject using avariety of pain scales.

Patient self-reporting can be used to assess whether pain is reduced;see, e.g., Katz and Melzack (1999) Surg. Clin. North Am. 79:231.Alternatively, an observational pain scale can be used. The LANSS PainScale can be used to assess whether pain is reduced; see, e.g., Bennett(2001) Pain 92:147. A visual analog pain scale can be used; see, e.g.,Schmader (2002) Clin. J. Pain 18:350. The Likert pain scale can be used;e.g., where 0 is no pain, 5 is moderate pain, and 10 is the worst painpossible. Self-report pain scales for children include, e.g., Faces PainScale; Wong-Baker FACES Pain Rating Scale; and Colored Analog Scale.Self-report pain scales for adults include, e.g., Visual Analog Scale;Verbal Numerical Rating Scale; Verbal Descriptor Scale; and Brief PainInventory. Pain measurement scales include, e.g., Alder Hey Triage PainScore (Stewart et al. (2004) Arch. Dis. Child. 89:625); Behavioral PainScale (Payen et al. (2001) Critical Care Medicine 29:2258); Brief PainInventory (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23:129);Checklist of Nonverbal Pain Indicators (Feldt (2000) Pain Manag. Nurs.1:13); Critical-Care Pain Observation Tool (Gelinas et al. (2006) Am. J.Crit. Care 15:420); COMFORT scale (Ambuel et al. (1992) J. PediatricPsychol. 17:95); Dallas Pain Questionnaire (Ozguler et al. (2002) Spine27:1783); Dolorimeter Pain Index (Hardy et al. (1952) Pain Sensationsand Reactions Baltimore: The Williams & Wilkins Co.); Faces PainScale—Revised (Hicks et al. (2001) Pain 93:173); Face Legs Activity CryConsolability Scale; McGill Pain Questionnaire (Melzack (1975) Pain1:277); Descriptor Differential Scale (Gracely and Kwilosz (1988) Pain35:279); Numerical 11 point Box (Jensen et al. (1989) Clin. J. Pain5:153); Numeric Rating Scale (Hartrick et al. (2003) Pain Pract. 3:310);Wong-Baker FACES Pain Rating Scale; and Visual Analog Scale (Huskisson(1982) 1 Rheumatol. 9:768).

In some cases, the light used to activate an opsin expressed in a neuron(e.g., a nociceptor) has an intensity of from about 0.05 mW/mm² to about0.1 mW/mm², from about 0.1 mW/mm² to about 0.2 mW/mm², from about 0.2mW/mm² to about 0.3 mW/mm², from about 0.3 mW/mm² to about 0.4 mW/mm²,from about 0.4 mW/mm² to about 0.5 mW/mm², from about 0.5 mW/mm² toabout 0.6 mW/mm², from about 0.6 mW/mm² to about 0.7 mW/mm², from aboutabout 0.7 mW/mm² to about 0.8 mW/mm², from about 0.8 mW/mm² to about 0.9mW/mm², or from about about 0.9 mW/mm² to about 1.0 mW/mm². In somecases, the light used to activate an opsin expressed in a neuron (e.g.,a nociceptor) has an intensity of from about 1.0 mW/mm² to about 1.1mW/mm², from about 1.1 mW/mm² to about 1.2 mW/mm², from about 1.2 mW/mm²to about 1.3 mW/mm², from 1.3 mW/mm² to about 1.4 mW/mm², from about 1.4mW/mm² to about 1.5 mW/mm², from about 1.5 mW/mm² to about 1.6 mW/mm²,from about 1.6 mW/mm² to about 1.7 mW/mm², from about 1.7 mW/mm² toabout 1.8 mW/mm², from about 1.8 mW/mm² to about 1.9 mW/mm², from about1.9 mW/mm² to about 2.0 mW/mm², from about 2.0 mW/mm² to about 2.5mW/mm², from about 2.5 mW/mm² to about 3 mW/mm², from about 3 mW/mm² toabout 3.5 mW/mm², from about 3.5 mW/mm² to about 4 mW/mm², from about 4mW/mm² to about 4.5 mW/mm², from about 4.5 mW/mm² to about 5 mW/mm²,from about 5 mW/mm² to about 5.5 mW/mm², from about 5.5 mW/mm² to about6 mW/mm², from about 6 mW/mm² to about 7 mW/mm², or from about 7 mW/mm²to about 10 mW/mm². In some cases, the light used to activate an opsinexpressed in a neuron (e.g., a nociceptor) has an intensity of fromabout 0.05 mW/mm² to about 0.1 mW/mm². In some cases, the light used toactivate an opsin expressed in a neuron (e.g., a nociceptor) has anintensity of about 0.25 mW/mm². In some cases, the light used toactivate an opsin expressed in a neuron (e.g., a nociceptor) has anintensity of about 1 mW/mm².

In some cases, the light is delivered transdermally or transcutaneously.In some cases, an implantable light source is used; and the light isdelivered to a site within the body. In some cases, the light isdelivered to a treatment site within the body. In some cases, the lightis delivered intracranially.

A nucleic acid comprising a nucleotide sequence encoding an opsin thatprovides for hyperpolarization of the cell in response to light of awavelength that activates the opsin can be introduced into a neuron(e.g., a nociceptor) by any convenient means. For example, a nucleicacid comprising a nucleotide sequence encoding an opsin that providesfor hyperpolarization of the cell in response to light of a wavelengththat activates the opsin can be introduced (e.g., injected) into a nervebundle or nerve fiber, such that the nucleic acid enters a neuron (e.g.,a nociceptor), where the opsin is produced in the neuron and is insertedinto the cell membrane. A nucleic acid comprising a nucleotide sequenceencoding an opsin that provides for hyperpolarization of the cell inresponse to light of a wavelength that activates the opsin can beintroduced (e.g., injected) proximal to a nerve. Stereotactic injectioncan be used; see, e.g., Stein et al., J. Virol, 73:34243429, 1999;Davidson et al., PNAS, 97:3428-3432, 2000; Davidson et al., Nat. Genet.3:219-223, 1993; and Alisky & Davidson, Hum. Gene Ther. 11:2315-2329,2000, the contents of each of which are hereby incorporated by referenceherein in their entireties.

A nucleic acid comprising a nucleotide sequence encoding an opsin thatprovides for hyperpolarization of the cell in response to light of awavelength that activates the opsin can be introduced (e.g., injected)intramuscularly. A nucleic acid comprising a nucleotide sequenceencoding an opsin that provides for hyperpolarization of the cell inresponse to light of a wavelength that activates the opsin can beadministered via any means, including, e.g., intravenous, intramuscular,intracranial, into a nerve, at or near a treatment site, and the like.Administration of an opsin-encoding nucleic acid can be carried out viainjection; via implantation at or near a treatment site of a compositioncomprising a nucleic acid encoding a light-activated polypeptide; via acatheter; or via any other means of delivery Administration of anopsin-encoding nucleic acid can be carried out via topical, intradermal,intravenous, intrathecal, or intrapleural administration. Administrationof an opsin-encoding nucleic acid can be carried out via intradermaladministration.

A light-activated protein (opsin) can be implanted into or proximal to anerve using a number of different methods. Example methods include, butare not limited to, the use of various delivery devices, such as gelatincapsules, liquid injections and the like. Such methods also include theuse of stereotactic surgery techniques such as frames or computerizedsurgical navigation systems to implant or otherwise access areas of thebody.

In some cases, a nucleic acid comprising a nucleotide sequence encodinga light-responsive opsin protein can be delivered directly to theneurons responsible for pain, where the delivery can be accomplishedwith a needle, catheter, or related device, using neurosurgicaltechniques known in the art, such as by stereotactic injection orfluoroscopy. Other methods to deliver a nucleic acid comprising anucleotide sequence encoding a light-responsive opsin protein to thenerves of interest can also be used, such as, but not limited to,transfection with ionic lipids or polymers, electroporation, opticaltransfection, impalefection, or via gene gun.

Hyperpolarizing Light-Responsive Polypeptides

As discussed above, methods for controlling pain according to thepresent disclosure generally involve introducing into a neuron of anindividual a nucleic acid comprising a nucleotide sequence encoding alight-activated polypeptide (an opsin) that provides forhyperpolarization of the cell in response to light of a wavelength thatactivates the light-activated polypeptide. A light-activated polypeptidecan be a polypeptide that allows one or more ions to pass through theplasma membrane of a target cell when the protein is illuminated withlight of an activating wavelength. Light-activated proteins may becharacterized as ion pump proteins, which facilitate the passage of asmall number of ions through the plasma membrane per photon of light, oras ion channel proteins, which allow a stream of ions to freely flowthrough the plasma membrane when the channel is open. Suitablelight-activated proteins for use in a subject method of reducing paininclude hyperpolarizing light-activated polypeptides.

Examples of suitable light-responsive polypeptides include, e.g., theHalorhodopsin family of light-responsive chloride pumps (e.g., NpHR,NpHR2.0, NpHR3.0, NpHR3.1). As another example, the GtR3 proton pump canbe used to promote neural cell membrane hyperpolarization in response tolight. As another example, eArch (a proton pump) can be used to promoteneural cell membrane hyperpolarization in response to light. As anotherexample, an ArchT opsin protein or a Mac opsin protein can be used topromote neural cell membrane hyperpolarization in response to light.

Enhanced Intracellular Transport Amino Acid Motifs

In some embodiments, the light-responsive opsin proteins expressed in acell can be fused to one or more amino acid sequence motifs selectedfrom the group consisting of a signal peptide, an ER export signal, amembrane trafficking signal, and/or an N-terminal golgi export signal.The one or more amino acid sequence motifs which enhancelight-responsive protein transport to the plasma membranes of mammaliancells can be fused to the N-terminus, the C-terminus, or to both the N-and C-terminal ends of the light-responsive protein. In some cases, theone or more amino acid sequence motifs which enhance light-responsiveprotein transport to the plasma membranes of mammalian cells is fusedinternally within a light-activated polypeptide. Optionally, thelight-responsive protein and the one or more amino acid sequence motifsmay be separated by a linker. In some embodiments, the light-responsiveprotein can be modified by the addition of a trafficking signal (ts)which enhances transport of the protein to the cell plasma membrane. Insome embodiments, the trafficking signal can be derived from the aminoacid sequence of the human inward rectifier potassium channel Kir2.1. Inother embodiments, the trafficking signal can comprise the amino acidsequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22).

Trafficking sequences that are suitable for use can comprise an aminoacid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, amino acid sequence identity to an amino acidsequence such a trafficking sequence of human inward rectifier potassiumchannel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)).

A trafficking sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Signal sequences that are suitable for use can comprise an amino acidsequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity to an amino acidsequence such as one of the following:

1) the signal peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ IDNO:23))

2) the β2 subunit signal peptide of the neuronal nicotinic acetylcholinereceptor (e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:24));

3) a nicotinic acetylcholine receptor signal sequence (e.g.,MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:25)); and

4) a nicotinic acetylcholine receptor signal sequence (e.g.,MRGTPLLLVVSLFSLLQD (SEQ ID NO:26)).

A signal sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

ER export sequences that are suitable for use in a modified opsininclude, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ ID NO:29);FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:31); and the like. An ER export sequence can have a length of fromabout 5 amino acids to about 25 amino acids, e.g., from about 5 aminoacids to about 10 amino acids, from about 10 amino acids to about 15amino acids, from about 15 amino acids to about 20 amino acids, or fromabout 20 amino acids to about 25 amino acids.

In some embodiments, the signal peptide sequence in the protein can bedeleted or substituted with a signal peptide sequence from a differentprotein.

Arch

In some embodiments, a suitable light-activated protein is anArchaerhodopsin (Arch) proton pump (e.g., a proton pump derived fromHalorubrum sodomense) that can transport one or more protons across theplasma membrane of a cell when the cell is illuminated with light. Thelight can have a wavelength between about 530 and about 595 nm or canhave a wavelength of about 560 nm. In some embodiments, the Arch proteincan comprise an amino acid sequence that is at least 75%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:1 (Arch). The Arch protein can additionallycomprise substitutions, deletions, and/or insertions introduced into anative amino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the Arch protein to transportions across the plasma membrane of a target cell. Additionally, the Archprotein can contain one or more conservative amino acid substitutionsand/or one or more non-conservative amino acid substitutions. The Archprotein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to transport ions across the plasma membrane of a target cell inresponse to light.

In some embodiments, an Arch protein comprises at least one (such asone, two, three, or more) amino acid sequence motifs that enhancetransport to the plasma membranes of target cells selected from thegroup consisting of a signal peptide, an ER export signal, and amembrane trafficking signal. In some embodiments, the Arch proteincomprises an N-terminal signal peptide and a C-terminal ER exportsignal. In some embodiments, the Arch protein comprises an N-terminalsignal peptide and a C-terminal trafficking signal. In some embodiments,the Arch protein comprises an N-terminal signal peptide, a C-terminal ERexport signal, and a C-terminal trafficking signal. In some embodiments,the Arch protein comprises a C-terminal ER export signal and aC-terminal trafficking signal. In some embodiments, the C-terminal ERexport signal and the C-terminal trafficking signal are linked by alinker. The linker can comprise any of about 5, 10, 20, 30, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker may further comprise a fluorescent protein, forexample, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments the ER export signal is more C-terminallylocated than the trafficking signal. In some embodiments the traffickingsignal is more C-terminally located than the ER Export signal.

In some embodiments, the trafficking signal can be derived from theamino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Traffickingsequences that are suitable for use can comprise an amino acid sequencehaving at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ERexport signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL(SEQ ID NO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ IDNO:29); FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV(SEQ ID NO:31); and the like.

ArchT

In some embodiments, a suitable light-activated protein is anArchaerhodopsin (ArchT) proton pump (e.g., a proton pump derived fromHalorubrum sp. TP009) that can transport one or more protons across theplasma membrane of a cell when the cell is illuminated with light. Thelight can have a wavelength between about 530 and about 595 nm or canhave a wavelength of about 560 nm. In some embodiments, the Arch proteincan comprise an amino acid sequence that is at least 75%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:3 (ArchT). The ArchT protein canadditionally comprise substitutions, deletions, and/or insertionsintroduced into a native amino acid sequence to increase or decreasesensitivity to light, increase or decrease sensitivity to particularwavelengths of light, and/or increase or decrease the ability of theArchT protein to transport ions across the plasma membrane of a targetcell. Additionally, the ArchT protein can contain one or moreconservative amino acid substitutions and/or one or morenon-conservative amino acid substitutions. The ArchT protein comprisingsubstitutions, deletions, and/or insertions introduced into the nativeamino acid sequence suitably retains the ability to transport ionsacross the plasma membrane of a target cell in response to light.

In some cases, the ArchT polypeptide comprises a membrane traffickingsignal and/or an ER export signal. In some embodiments, the traffickingsignal can be derived from the amino acid sequence of the human inwardrectifier potassium channel Kir2.1. In other embodiments, thetrafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Trafficking sequences that aresuitable for use can comprise an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence such a traffickingsequence of human inward rectifier potassium channel Kir2.1 (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ER exportsignal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ ID NO:29);FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:31); and the like.

GtR3

In some embodiments, the light-responsive proton pump protein can beresponsive to blue light and can be derived from Guillardia theta,wherein the proton pump protein can be capable of mediating ahyperpolarizing current in the cell when the cell is illuminated withblue light. The light can have a wavelength between about 450 and about495 nm or can have a wavelength of about 490 nm. In another embodiment,the light-responsive proton pump protein can comprise an amino acidsequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the sequence shown in SEQ ID NO:4 (GtR3). Thelight-responsive proton pump protein can additionally comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive protonpump protein to regulate the polarization state of the plasma membraneof the cell. Additionally, the light-responsive proton pump protein cancontain one or more conservative amino acid substitutions and/or one ormore non-conservative amino acid substitutions. The light-responsiveproton pump protein comprising substitutions, deletions, and/orinsertions introduced into the native amino acid sequence suitablyretains the ability to hyperpolarize the plasma membrane of a neuronalcell in response to light.

In other aspects of the methods disclosed herein, the light-responsiveproton pump protein can comprise a core amino acid sequence at least75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:4 and at least one (such asone, two, three, or more) amino acid sequence motifs which enhancetransport to the plasma membranes of mammalian cells selected from thegroup consisting of a signal peptide, an ER export signal, and amembrane trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal ER export signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide, a C-terminalER Export signal, and a C-terminal trafficking signal. In someembodiments, the light-responsive proton pump protein comprises aC-terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 400, or 500 amino acids in length. The linker may furthercomprise a fluorescent protein, for example, but not limited to, ayellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER Export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

In some embodiments, the trafficking signal can be derived from theamino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Traffickingsequences that are suitable for use can comprise an amino acid sequencehaving at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ERexport signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL(SEQ ID NO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ IDNO:29); FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV(SEQ ID NO:31); and the like.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive proton pump proteins described herein, such as alight-responsive proton pump protein comprising a core amino acidsequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:4. Alsoprovided herein are expression vectors (such as a viral vector describedherein) comprising a polynucleotide encoding the proteins describedherein, such as a light-responsive proton pump protein comprising a coreamino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:4.

Oxy

In some embodiments, a light-activated protein is an Oxyrrhis marina(Oxy) proton pump that can transport one or more protons across theplasma membrane of a cell when the cell is illuminated with light. Thelight can have a wavelength between about 500 and about 560 nm or canhave a wavelength of about 530 nm. In some embodiments, the Oxy proteincan comprise an amino acid sequence that is at least 75%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:5. The Oxy protein can additionally comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the Oxy protein to transportions across the plasma membrane of a target cell. Additionally, the Oxyprotein can contain one or more conservative amino acid substitutionsand/or one or more non-conservative amino acid substitutions. The Oxyprotein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to transport ions across the plasma membrane of a target cell inresponse to light.

In some embodiments, an Oxy protein comprises at least one (such as one,two, three, or more) amino acid sequence motifs that enhance transportto the plasma membranes of target cells selected from the groupconsisting of a signal peptide, an ER export signal, and a membranetrafficking signal. In some embodiments, the Oxy protein comprises anN-terminal signal peptide and a C-terminal ER export signal. In someembodiments, the Oxy protein comprises an N-terminal signal peptide anda C-terminal trafficking signal. In some embodiments, the Oxy proteincomprises an N-terminal signal peptide, a C-terminal ER export signal,and a C-terminal trafficking signal. In some embodiments, the Oxyprotein comprises a C-terminal ER export signal and a C-terminaltrafficking signal. In some embodiments, the C-terminal ER export signaland the C-terminal trafficking signal are linked by a linker. The linkercan comprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linkermay further comprise a fluorescent protein, for example, but not limitedto, a yellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

Mac

In some embodiments, the light-responsive proton pump protein can beresponsive to light and can be derived from Leptosphaeria maculans,wherein the proton pump protein can be capable of pumping protons acrossthe membrane of a cell when the cell is illuminated with 520 nm to 560nm light. The light can have a wavelength between about 520 nm to about560 nm. In another embodiment, the light-responsive proton pump proteincan comprise an amino acid sequence at least 75%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceshown in SEQ ID NO:6 or SEQ ID NO:7 (Mac; Mac 3.0). The light-responsiveproton pump protein can additionally comprise substitutions, deletions,and/or insertions introduced into a native amino acid sequence toincrease or decrease sensitivity to light, increase or decreasesensitivity to particular wavelengths of light, and/or increase ordecrease the ability of the light-responsive proton pump protein toregulate the polarization state of the plasma membrane of the cell.Additionally, the light-responsive proton pump protein can contain oneor more conservative amino acid substitutions and/or one or morenon-conservative amino acid substitutions. The light-responsive protonpump protein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to pump protons across the plasma membrane of a neuronal cell inresponse to light.

In other aspects of the methods disclosed herein, the light-responsiveproton pump protein can comprise a core amino acid sequence at least75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:6 and at least one (such asone, two, three, or more) amino acid sequence motifs which enhancetransport to the plasma membranes of mammalian cells selected from thegroup consisting of a signal peptide, an ER export signal, and amembrane trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal ER export signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide, a C-terminalER Export signal, and a C-terminal trafficking signal. In someembodiments, the light-responsive proton pump protein comprises aC-terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 400, or 500 amino acids in length. The linker may furthercomprise a fluorescent protein, for example, but not limited to, ayellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER Export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

In some embodiments, the trafficking signal can be derived from theamino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Traffickingsequences that are suitable for use can comprise an amino acid sequencehaving at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ERexport signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL(SEQ ID NO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ IDNO:29); FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV(SEQ ID NO:31); and the like.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive proton pump proteins described herein, such as alight-responsive proton pump protein comprising a core amino acidsequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:6. Alsoprovided herein are expression vectors (such as a viral vector describedherein) comprising a polynucleotide encoding the proteins describedherein, such as a light-responsive proton pump protein comprising a coreamino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:6.

Further disclosure related to light-activated proton pump proteins canbe found in International Patent Application No. PCT/US2011/028893, thedisclosure of which is hereby incorporated by reference in its entirety.

NpHR

In some cases, a suitable light-responsive chloride pump proteinsexpressed on the plasma membranes of the neurons described above can bederived from Natronomonas pharaonis. In some embodiments, thelight-responsive chloride pump proteins can be responsive to amber lightas well as red light and can mediate a hyperpolarizing current in theneuron when the light-responsive chloride pump proteins are illuminatedwith amber or red light. The wavelength of light which can activate thelight-responsive chloride pumps can be between about 580 and 630 nm. Insome embodiments, the light can be at a wavelength of about 589 nm orthe light can have a wavelength greater than about 630 nm (e.g. lessthan about 740 nm). In another embodiment, the light has a wavelength ofaround 630 nm. In some embodiments, the light-responsive chloride pumpprotein can hyperpolarize a neural membrane for at least about 90minutes when exposed to a continuous pulse of light. In someembodiments, the light-responsive chloride pump protein can comprise anamino acid sequence at least about 75%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQID NO:16. Additionally, the light-responsive chloride pump protein cancomprise substitutions, deletions, and/or insertions introduced into anative amino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive proteinto regulate the polarization state of the plasma membrane of the cell.In some embodiments, the light-responsive chloride pump protein containsone or more conservative amino acid substitutions. In some embodiments,the light-responsive protein contains one or more non-conservative aminoacid substitutions. The light-responsive protein comprisingsubstitutions, deletions, and/or insertions introduced into the nativeamino acid sequence suitably retains the ability to hyperpolarize theplasma membrane of a neuronal cell in response to light.

Additionally, in other aspects, the light-responsive chloride pumpprotein can comprise a core amino acid sequence at least about 75%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe sequence shown in SEQ ID NO:16 and an endoplasmic reticulum (ER)export signal. This ER export signal can be fused to the C-terminus ofthe core amino acid sequence or can be fused to the N-terminus of thecore amino acid sequence. In some embodiments, the ER export signal islinked to the core amino acid sequence by a linker. The linker cancomprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linkermay further comprise a fluorescent protein, for example, but not limitedto, a yellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodiments,the ER export signal can comprise the amino acid sequence FXYENE (SEQ IDNO:30), where X can be any amino acid. In another embodiment, the ERexport signal can comprise the amino acid sequence VXXSL, where X can beany amino acid. In some embodiments, the ER export signal can comprisethe amino acid sequence FCYENEV (SEQ ID NO:31).

Endoplasmic reticulum (ER) export sequences that are suitable for use ina modified opsin of the present disclosure include, e.g., VXXSL (where Xis any amino acid) (e.g., VKESL (SEQ ID NO:27); VLGSL (SEQ ID NO:28);etc.); NANSFCYENEVALTSK (SEQ ID NO:29); FXYENE (where X is any aminoacid) (SEQ ID NO:30), e.g., FCYENEV (SEQ ID NO:31); and the like. An ERexport sequence can have a length of from about 5 amino acids to about25 amino acids, e.g., from about 5 amino acids to about 10 amino acids,from about 10 amino acids to about 15 amino acids, from about 15 aminoacids to about 20 amino acids, or from about 20 amino acids to about 25amino acids.

In other aspects, the light-responsive chloride pump proteins describedherein can comprise a light-responsive protein expressed on the cellmembrane, wherein the protein comprises a core amino acid sequence atleast 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the sequence shown in SEQ ID NO:16 and a traffickingsignal (e.g., which can enhance transport of the light-responsivechloride pump protein to the plasma membrane). The trafficking signalmay be fused to the C-terminus of the core amino acid sequence or may befused to the N-terminus of the core amino acid sequence. In someembodiments, the trafficking signal can be linked to the core amino acidsequence by a linker which can comprise any of about 5, 10, 20, 30, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 aminoacids in length. The linker may further comprise a fluorescent protein,for example, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the trafficking signal can be derived fromthe amino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22).

In some aspects, the light-responsive chloride pump protein can comprisea core amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQID NO:16 and at least one (such as one, two, three, or more) amino acidsequence motifs which enhance transport to the plasma membranes ofmammalian cells selected from the group consisting of an ER exportsignal, a signal peptide, and a membrane trafficking signal. In someembodiments, the light-responsive chloride pump protein comprises anN-terminal signal peptide, a C-terminal ER Export signal, and aC-terminal trafficking signal. In some embodiments, the C-terminal ERExport signal and the C-terminal trafficking signal can be linked by alinker. The linker can comprise any of about 5, 10, 20, 30, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker can also further comprise a fluorescent protein, forexample, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments the ER Export signal can be moreC-terminally located than the trafficking signal. In other embodimentsthe trafficking signal is more C-terminally located than the ER Exportsignal. In some embodiments, the signal peptide comprises the amino acidsequence MTETLPPVTESAVALQAE (SEQ ID NO:32). In another embodiment, thelight-responsive chloride pump protein comprises an amino acid sequenceat least 95% identical to SEQ ID NO:17.

Moreover, in other aspects, the light-responsive chloride pump proteinscan comprise a core amino acid sequence at least 75%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:16, wherein the N-terminal signal peptide ofSEQ ID NO:16 is deleted or substituted. In some embodiments, othersignal peptides (such as signal peptides from other opsins) can be used.The light-responsive protein can further comprise an ER transport signaland/or a membrane trafficking signal described herein. In someembodiments, the light-responsive chloride pump protein comprises anamino acid sequence at least 95% identical to SEQ ID NO:18.

In some embodiments, the light-responsive opsin protein is a NpHR opsinprotein comprising an amino acid sequence at least 75%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical to the sequence shown in SEQ ID NO:16. Insome embodiments, the NpHR opsin protein further comprises anendoplasmic reticulum (ER) export signal and/or a membrane traffickingsignal. For example, the NpHR opsin protein comprises an amino acidsequence at least 95% identical to the sequence shown in SEQ ID NO:16and an endoplasmic reticulum (ER) export signal. In some embodiments,the amino acid sequence at least 95% identical to the sequence shown inSEQ ID NO:16 is linked to the ER export signal through a linker. In someembodiments, the ER export signal comprises the amino acid sequenceFXYENE (SEQ ID NO:30), where X can be any amino acid. In anotherembodiment, the ER export signal comprises the amino acid sequenceVXXSL, where X can be any amino acid. In some embodiments, the ER exportsignal comprises the amino acid sequence FCYENEV (SEQ ID NO:31). In someembodiments, the NpHR opsin protein comprises an amino acid sequence atleast 95% identical to the sequence shown in SEQ ID NO:16, an ER exportsignal, and a membrane trafficking signal. In other embodiments, theNpHR opsin protein comprises, from the N-terminus to the C-terminus, theamino acid sequence at least 95% identical to the sequence shown in SEQID NO:16, the ER export signal, and the membrane trafficking signal. Inother embodiments, the NpHR opsin protein comprises, from the N-terminusto the C-terminus, the amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO:16, the membrane trafficking signal, and theER export signal. In some embodiments, the membrane trafficking signalis derived from the amino acid sequence of the human inward rectifierpotassium channel Kir2.1. In some embodiments, the membrane traffickingsignal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ IDNO:22). In some embodiments, the membrane trafficking signal is linkedto the amino acid sequence at least 95% identical to the sequence shownin SEQ ID NO:16 by a linker. In some embodiments, the membranetrafficking signal is linked to the ER export signal through a linker.The linker may comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. Thelinker may further comprise a fluorescent protein, for example, but notlimited to, a yellow fluorescent protein, a red fluorescent protein, agreen fluorescent protein, or a cyan fluorescent protein. In someembodiments, the light-responsive opsin protein further comprises anN-terminal signal peptide. In some embodiments, the light-responsiveopsin protein comprises the amino acid sequence of SEQ ID NO:17. In someembodiments, the light-responsive opsin protein comprises the amino acidsequence of SEQ ID NO:18.

Also provided herein are polynucleotides encoding any of thelight-responsive chloride ion pump proteins described herein, such as alight-responsive protein comprising a core amino acid sequence at least75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:16, an ER export signal,and a membrane trafficking signal. In another embodiment, thepolynucleotides comprise a sequence which encodes an amino acid at least95% identical to SEQ ID NO:17 and SEQ ID NO:18. The polynucleotides maybe in an expression vector (such as, but not limited to, a viral vectordescribed herein). The polynucleotides may be used for expression of thelight-responsive chloride ion pump proteins.

Further disclosure related to light-responsive chloride pump proteinscan be found in U.S. Patent Application Publication Nos: 2009/0093403and 2010/0145418 as well as in International Patent Application No:PCT/US2011/028893, the disclosures of each of which are herebyincorporated by reference in their entireties.

Dunaliella salina Light-Activated Polypeptide

In some embodiments, a suitable light-responsive ion channel protein canbe responsive to 470 nm-510 nm light and can be derived from Dunaliellasalina, wherein the ion channel protein can be capable of mediating ahyperpolarizing current in the cell when the cell is illuminated withlight. The light can have a wavelength between about 470 nm and about510 nm or can have a wavelength of about 490 nm. In some embodiments,the light-responsive ion channel protein can comprise an amino acidsequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:15. Thelight-responsive ion channel protein can additionally comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive ionchannel protein to regulate the polarization state of the plasmamembrane of the cell. Additionally, the light-responsive ion channelprotein can contain one or more conservative amino acid substitutionsand/or one or more non-conservative amino acid substitutions. Thelight-responsive ion channel protein comprising substitutions,deletions, and/or insertions introduced into the native amino acidsequence suitably retains the ability to transport ions across theplasma membrane of a neuronal cell in response to light.

In other aspects of the methods disclosed herein, the light-responsiveion channel protein can comprise a core amino acid sequence at least75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:15 and at least one (suchas one, two, three, or more) amino acid sequence motifs which enhancetransport to the plasma membranes of mammalian cells selected from thegroup consisting of a signal peptide, an ER export signal, and amembrane trafficking signal. In some embodiments, the light-responsiveproton ion channel comprises an N-terminal signal peptide and aC-terminal ER export signal. In some embodiments, the light-responsiveion channel protein comprises an N-terminal signal peptide and aC-terminal trafficking signal. In some embodiments, the light-responsiveion channel protein comprises an N-terminal signal peptide, a C-terminalER Export signal, and a C-terminal trafficking signal. In someembodiments, the light-responsive ion channel protein comprises aC-terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 400, or 500 amino acids in length. The linker may furthercomprise a fluorescent protein, for example, but not limited to, ayellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER Export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive channel proteins described herein, such as alight-responsive ion channel protein comprising a core amino acidsequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the sequence shown in SEQ ID NO:15. Also providedherein are expression vectors (such as a viral vector described herein)comprising a polynucleotide encoding the proteins described herein, suchas a light-responsive channel protein comprising a core amino acidsequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the sequence shown in SEQ ID NO:15.

Polynucleotides and Vectors

As discussed above, methods for controlling pain according to thepresent disclosure generally involve introducing into a neuron (e.g., anociceptor) of an individual a nucleic acid comprising a nucleotidesequence encoding an opsin that provides for hyperpolarization of thecell in response to light of a wavelength that activates the opsin.Suitable nucleic acids comprise a nucleotide sequence that encodes oneor more of the light-activated polypeptides (opsins) described herein(e.g., one or more light-activated polypeptides as described herein). Insome embodiments, a polynucleotide comprises an expression cassette,wherein the expression cassette contains a plurality of components(e.g., coding sequences; transcription control sequences; etc.) that areutilized to express one or more proteins encoded by the polynucleotidein a target cell.

In some embodiments, a portion of a polynucleotide encoding alight-activated polypeptide is operably linked to a promoter sequence.Any suitable promoter that functions in a target cell can be used forexpression of a polynucleotide encoding a light-activated polypeptide.In certain embodiments, a promoter sequence can be a promoter that isspecific to a particular target cell type or to a particular tissuetype, such as a particular neuron or a pan-neuronal promoter. Initiationcontrol regions of promoters, which are useful to drive expression ofpolynucleotides in a specific animal cell, are numerous and familiar tothose skilled in the art. Virtually any promoter capable of drivingexpression of the subject polynucleotides can be used. In someembodiments, the promoter used to drive expression of a subject proteincan be the Thy1 promoter (See, e.g., Llewellyn, et al., 2010, Nat. Med.,16(10):1161-1166). In some embodiments, the promoter used to driveexpression of a subject protein can be a human synapsin (hSyn) promoter,a human elongation factor 1-α (EF1α) promoter, a cytomegalovirus (CMV)promoter, a CMV early enhancer/chicken (3 actin (CAG) promoter, asynapsin-I promoter (e.g., a human synapsin-I promoter), a humansynuclein 1 promoter, a human Thy1 promoter, acalcium/calmodulin-dependent kinase II alpha (CAMKIIα) promoter, or anyother promoter capable of driving expression of the a subject nucleicacid sequence in a target cell.

Neuron-specific promoters and other control elements (e.g., enhancers)are known in the art, and can be operably linked to an opsin-encodingnucleotide sequence. Suitable neuron-specific control sequences include,but are not limited to, a neuron-specific enolase (NSE) promoter (see,e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. Nos. 6,649,811,5,387,742); an aromatic amino acid decarboxylase (AADC) promoter; aneurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsinpromoter (see, e.g., GenBank HUMSYN1B, M55301); a thy-1 promoter (see,e.g., Chen et al. (1987) Cell 51:7-19); a serotonin receptor promoter(see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see,e.g., Nucl. Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594(1991)); a GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad.Sci. USA 88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick etal., Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge etal., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalinpromoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelinbasic protein (MBP) promoter; a CMV enhancer/platelet-derived growthfactor-β promoter (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60);a motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Pat. No.7,632,679; and Lee et al. (2004) Development 131:3295-3306); and analpha subunit of Ca(²⁺)-calmodulin-dependent protein kinase I1 (CaMK1Iα)promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA93:13250).

In some embodiments, a promoter may be an inducible promoter. Forexample, the promoter may be induced by a trans-acting factor thatresponds to an exogenously administered drug. Examples of induciblepromoters include, but are not limited to, tetracycline-on ortetracycline-off promoters, or tamoxifen-inducible CreER.

In some embodiments, a subject polynucleotide may comprise a ribosomalskip sequence that can be used to generate two separate proteins fromthe same transcript. In such embodiments, a subject polynucleotide willtypically include a coding sequence that encodes a light-activatedprotein as well as a response protein. In these embodiments, a ribosomalskip sequence may be placed between the two coding sequences to producetwo distinct proteins (namely, the light-activated protein and theresponse protein) from the same transcript.

As noted above, in some cases, the nucleic acid is a recombinantexpression vector comprising a nucleotide sequence encoding alight-activated polypeptide or any variant thereof as described herein.Suitable expression vectors include vectors comprising a nucleotidesequence that encodes an RNA (e.g., an mRNA) that when transcribed fromthe polynucleotides of the vector will result in the accumulation of asubject protein on the plasma membranes of target cells. Vectors whichmay be used include, without limitation, lentiviral, herpes simplexvirus, adenoviral, and adeno-associated virus (AAV) vectors. Lentiviralvectors include, but are not limited to human immunodeficiency virus(HIV)-based vectors. Lentiviral vectors may be pseudotyped with theenvelope proteins of other viruses, including, but not limited tovesicular stomatitis virus (VSV), rabies, Mo-murine leukemia virus(MLV), baculovirus and Ebola. Such vectors may be prepared usingstandard methods in the art.

In some embodiments, a vector may be a recombinant AAV vector. AAVvectors are DNA viruses of relatively small size that can integrate, ina stable and site-specific manner, into the genome of the cells thatthey infect. They are able to infect a wide spectrum of cells withoutinducing any effects on cellular growth, morphology or differentiation,and they do not appear to be involved in human pathologies. The AAVgenome has been cloned, sequenced and characterized. It encompassesapproximately 4700 bases and contains an inverted terminal repeat (ITR)region of approximately 145 bases at each end, which serves as an originof replication for the virus. The remainder of the genome is dividedinto two essential regions that carry the encapsidation functions: theleft-hand part of the genome that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome that contains the cap gene encoding the capsidproteins of the virus.

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (see, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” In Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 5-14,Hudder Arnold, London, U K (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p 15-23, Hudder Arnold, London, UK (2006),the disclosures of each of which are hereby incorporated by referenceherein in their entireties). Methods for purifying for vectors may befound in, for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and6,995,006 and WO/1999/011764 titled “Methods for Generating High TiterHelper-free Preparation of Recombinant AAV Vectors”, the disclosures ofwhich are herein incorporated by reference in their entirety. Methods ofpreparing AAV vectors in a baculovirus system are described in, e.g., WO2008/024998. AAV vectors can be self-complementary or single-stranded.Preparation of hybrid vectors is described in, for example, PCTApplication No. PCT/US2005/027091, the disclosure of which is hereinincorporated by reference in its entirety. The use of vectors derivedfrom the AAVs for transferring genes in vitro and in vivo has beendescribed (See e.g., International Patent Application Publication Nos.:91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and5,139,941; and European Patent No.: 0488528, all of which are herebyincorporated by reference herein in their entireties). Thesepublications describe various AAV-derived constructs in which the repand/or cap genes are deleted and replaced by a gene of interest, and theuse of these constructs for transferring the gene of interest in vitro(into cultured cells) or in vivo (directly into an organism). Thereplication-defective recombinant AAVs according to the presentdisclosure can be prepared by co-transfecting a plasmid containing thenucleic acid sequence of interest flanked by two AAV inverted terminalrepeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes(rep and cap genes), into a cell line that is infected with a humanhelper virus (for example an adenovirus). The AAV recombinants that areproduced are then purified by standard techniques.

In some embodiments, the vector(s) for use in the methods of the presentdisclosure are encapsidated into a virus particle (e.g. AAV virusparticle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, andAAV16). Accordingly, the present disclosure includes a recombinant virusparticle (recombinant because it contains a recombinant polynucleotide)comprising any of the vectors described herein. Methods of producingsuch particles are known in the art and are described in U.S. Pat. No.6,596,535, the disclosure of which is hereby incorporated by referencein its entirety. In some cases, AAV6 is used. In some cases, AAV1 isused.

Pharmaceutical Compositions

Aspects of the present disclosure include pharmaceutical compositionsthat polynucleotides, vectors, or components thereof, described above.The subject pharmaceutical compositions may be administered to a subjectfor purposes of genetically modifying a target cell so that the targetcell expresses one or more light-activated proteins. A subjectpharmaceutical composition may, in some embodiments, comprise apharmaceutically acceptable excipient. In some embodiments, apharmaceutical composition may comprise components to facilitatedelivery of the subject polynucleotides or vectors to a target cell,including but not limited to transfection reagents or componentsthereof, such as lipids, polymers, and the like.

In some embodiments, a subject pharmaceutical composition will besuitable for injection into a subject, e.g., will be sterile. Forexample, in some embodiments, a subject pharmaceutical composition willbe suitable for injection into a subject, e.g., where the composition issterile and is free of detectable pyrogens and/or other toxins.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public as well, andmay be incorporated into the pharmaceutical compositions of the presentdisclosure without limitation.

Delivery Devices

In some cases, a delivery device is used to deliver a nucleic acidencoding a light-activated polypeptide, or a pharmaceutical compositioncomprising same, to a target cell. The delivery device may provideregular, irregular, programmed, or clinician- or patient-activated dosesof the nucleic acid or pharmaceutical composition to one or more targetcells to ensure that the target cells continue to express the encodedlight-activated polypeptide.

Suitable delivery devices may generally include one or more components,such as reservoirs, pumps, actuators, tubing components, needles,catheters, and any other suitable components for delivering the nucleicacid or pharmaceutical composition to a target cell or tissue of anindividual. Delivery devices may also include components that facilitatecomputerized operation, such as a power source, a processor comprising amemory, a user input device, and/or a graphical user interface. In someembodiments, a delivery device may be completely or partiallyimplantable within a patient. In some embodiments, a delivery device maybe operated by a caregiver, wherein the device is introduced into aportion of the patient's body, e.g., into the patient's brain, and asubject pharmaceutical composition is delivered to a target tissue,e.g., a portion of the patient's brain. In some embodiments, followingdelivery of the pharmaceutical composition, the device may be removed.In other embodiments, the device may be kept in place for later deliveryof additional pharmaceutical compositions.

Light-Generating Devices

In carrying out a subject method of controlling pain, a light-generatingdevice can be used to deliver light to target cells that express one ormore light-activated polypeptides. Light-generating devices suitable foruse with a method of the present disclosure can generally produce lightof a variety of different wavelengths from one or more light sources onthe device. In some embodiments, a light-generating device may include alight cuff or sleeve that can be placed around or near target cellsexpressing one or more subject proteins. In some embodiments, a portionof the light source or the entire light source may be implantable. Thesubject light-generating devices may be of any useful configuration forstimulating the light-activated proteins disclosed herein. In someembodiments, for example, a light-generating device may comprisecomponents that facilitate exclusive illumination of a target cell ortissue. For example, in some embodiments, a light-generating device mayexclusively direct light to a target cell, a portion of a target cell,e.g., a particular axon of a nerve cell, or a specific anatomicalstructure, such as, e.g. a bundle of nerve fibers, a target tissue, or aportion of the spinal cord. By “exclusively direct light” is meant thatthe light-generating device only delivers light to the specific targetstructure, and does not illuminate other structures. For examples, insome embodiments, a light-generating device may be configured toilluminate an axon of a nerve cell, but not illuminate any other portionof the nerve cell. In this way, the light from the Light-generatingdevice only affects light-activated proteins in the specific targetstructure that is illuminated.

In some embodiments, a light-generating device may not completelysurround the region containing a target cell expressing alight-activated protein, but, rather, can have a U-shape. In someembodiments, a light-generating device can have an attachment arm thatcan be used to guide the light-generating device to a specific region ortarget structure, e.g., a specific neuronal region. The attachment armcan be removed following implantation of the light-generating device orcan be left in place to fix the position of the light-generating devicein proximity to the target cells of interest.

In some embodiments, the subject light-generating devices may comprisean inner body, the inner body having at least one means for generatinglight which is connected to a power source. In some embodiments, thepower source can be an internal battery for powering theLight-generating device. In some embodiments, an implantablelight-generating device may comprise an external antenna for receivingwirelessly transmitted electromagnetic energy from an external sourcefor powering device. The wirelessly transmitted electromagnetic energycan be a radio wave, a microwave, or any other electromagnetic energysource that can be transmitted from an external source to power thelight-generating device. In some embodiments, the light-generatingdevice is controlled by, e.g., an integrated circuit produced usingsemiconductor or other processes known in the art.

In some embodiments, the light-generating device may comprise a lightemitting diode (LED). In some embodiments, the LED can generate blueand/or green light. In other embodiments, the LED can generate amberand/or yellow light. In some embodiments, several micro LEDs areembedded into the inner body of the light-generating device. In otherembodiments, the light-generating device is a solid state laser diode orany other means capable of generating light. The light-generating devicecan generate light having a wavelength and intensity sufficient toactivate a subject light-activated protein. In some embodiments, alight-generating device produces light having an intensity of any ofabout 0.05 mW/mm², 0.1 mW/mm², 0.2 mW/mm², 0.3 mW/mm², 0.4 mW/mm², 0.5mW/mm², about 0.6 mW/mm², about 0.7 mW/mm², about 0.8 mW/mm², about 0.9mW/mm², about 1.0 mW/mm², about 1.1 mW/mm², about 1.2 mW/mm², about 1.3mW/mm², about 1.4 mW/mm², about 1.5 mW/mm², about 1.6 mW/mm², about 1.7mW/mm², about 1.8 mW/mm², about 1.9 mW/mm², about 2.0 mW/mm², about 2.1mW/mm², about 2.2 mW/mm², about 2.3 mW/mm², about 2.4 mW/mm², about 2.5mW/mm², about 3 mW/mm², about 3.5 mW/mm², about 4 mW/mm², about 4.5mW/mm², about 5 mW/mm², about 5.5 mW/mm², about 6 mW/mm², about 7mW/mm², about 8 mW/mm², about 9 mW/mm², or about 10 mW/mm², inclusive,including values in between these numbers. In some embodiments, thelight-generating device produces light having an intensity of at leastabout 10 Hz, such as up to about 25 Hz, such as up to about 50 Hz, suchas up to about 75 Hz, such as up to about 100 Hz.

Suitable light-generating devices are generally capable of generatinglight having a wavelength ranging from about 350 nm, up to about 360 nm,up to about 370 nm, up to about 380 nm, up to about 390 nm, up to about400 nm, up to about 410 nm, up to about 420 nm, up to about 430 nm, upto about 440 nm, up to about 450 nm, up to about 460 nm, up to about 470nm, up to about 480 nm, up to about 490 nm, up to about 500 nm, up toabout 510 nm, up to about 520 nm, up to about 530 nm, up to about 540nm, up to about 550 nm, up to about 560 nm, up to about 570 nm, up toabout 580 nm, up to about 590 nm, up to about 600 nm, up to about 610nm, up to about 620 nm, up to about 630 nm, up to about 640 nm, up toabout 650 nm, up to about 660 nm, up to about 670 nm, up to about 680nm, up to about 690 nm, up to about 700 nm, up to about 710 nm, up toabout 720 nm, up to about 730 nm, up to about 740 nm, and/or up to about750 nm.

In some embodiments, a suitable light-generating device may include oneor more optical fibers that can transmit light from a light source anddeliver the light to a target structure. The optical fibers may compriseplastic or glass materials, and in some embodiments may be suitablyflexible to facilitate placement of the light-generating device inlocations that could not be accommodated by rigid structures. Forexample, in some embodiments, a light-generating device may comprise alight source that generates light, as well as one or more optical fibersthat can be placed in various locations on or in the patient's body.Light from the light source can pass through the optical fiber, passingaround corners and bends in the optical fiber, and emerge at the end ofthe optical fiber to deliver light to a target structure.

In some embodiments, a suitable light-generating device may comprise aplurality of light sources that can be used to illuminate a targettissue with different wavelengths of light. For example, in someembodiments, a light-generating device may comprise a first light sourcethat generates light of a first wavelength, e.g., red light, and asecond light source that generates light of a second wavelength, e.g.,green light. Such light-generating devices may be used to simultaneouslyilluminate the same target tissue with light of both wavelengths, or mayalternately illuminate the target tissue with light of the firstwavelength and light of the second wavelength. In some embodiments, suchlight generating devices may be used to deliver light from the samelight source different target tissues. For example, in some embodimentsa light-generating device may deliver light of a first wavelength to afirst target tissue, and may deliver light of a second wavelength to adifferent target tissue.

Control Devices

In some cases, a control device that can control, or modulate, theamount of light that is emitted from the light-generating device is usedin a subject method. In some embodiments, a control device may beconfigured to modulate the wavelength and/or the intensity of light thatis delivered to a target tissue from a light-generating device. In someembodiments, a control device may be configured to modulate thefrequency and/or duration of light that is delivered to a target tissuefrom a light-generating device. For example, in some embodiments, acontrol device may be configured to deliver pulses of light from thelight-generating device to a target tissue. The control device canmodulate the frequency and/or duration of the light pulses such that thetarget tissue is illuminated with light from the light-generatingdevice, e.g., at a regular or irregular rate, according to a user input,etc. In some embodiments, a control device can produce pulses of lightfrom the light-generating device that have a duration ranging from about1 millisecond or less, up to about 1 second, up to about 10 seconds, upto about 20 seconds, up to about 30 seconds, up to about 40 seconds, upto about 50 seconds, up to about 60 seconds or more. In someembodiments, a control device can produce pulses of light from thelight-generating device that have a frequency of 1 pulse permillisecond, up to about 1 pulse per second, up about 1 pulse perminute, up to about 1 pulse per 10 minutes, up to about 1 pulse per 20minutes, up to about 1 pulse per 30 minutes.

In some embodiments, a suitable control device may comprise a powersource that can be mounted to a wireless transmitter. In someembodiments, a suitable control device may comprise a power source thatcan be mounted to a transmitting coil. In some embodiments, a batterycan be connected to the power source for providing power thereto. Aswitch can be connected to the power source, allowing an operator (e.g.,a patient or caregiver) to manually activate or deactivate the powersource. In some embodiments, upon activation of the switch, the powersource can provide power to the light-generating device throughelectromagnetic coupling between the transmitting coil on the controldevice and an antenna (which may be an external antenna or an internalantenna) of an implantable light-generating device (such as a light cuffor sleeve). The transmitting coil can establish an electromagneticcoupling with the external antenna of the implantable light-generatingdevice when in proximity thereof, for supplying power to thelight-generating device and for transmitting one or more control signalsto the light-generating device. In some embodiments, the electromagneticcoupling between the transmitting coil of the control device and theexternal antenna of the implantable light-generating device can beradio-frequency magnetic inductance coupling. When radio-frequencymagnetic inductance coupling is used, the operational frequency of theradio wave can be between about 1 and 20 MHz, inclusive, including anyvalues in between these numbers (for example, about 1 MHz, about 2 MHz,about 3 MHz, about 4 MHz, about 5 MHz, about 6 MHz, about 7 MHz, about 8MHz, about 9 MHz, about 10 MHz, about 11 MHz, about 12 MHz, about 13MHz, about 14 MHz, about 15 MHz, about 16 MHz, about 17 MHz, about 18MHz, about 19 MHz, or about 20 MHz). In some cases, the operationalfrequency of the radio wave can be between about 20 MHz and 5 GHz, e.g.,from about 20 MHz to about 50 MHz, from about 50 MHz to about 250 MHz,from about 250 MHz to about 500 MHz, from about 500 MHz to about 750MHz, from about 750 MHz to about 1 GHz, from about 1 GHz to about 2 GHz,from about 2 GHz to about 3 GHz, from about 3 GHz to about 4 GHz, orfrom about 4 GHz to about 5 GHz. For example, where midfieldradiofrequency coupling is used, the operational frequency can be from690 MHz to 2.2 GHz. Other coupling techniques may be used, such as anoptical receiver, infrared, or a biomedical telemetry system (See, e.g.,Kiourti, “Biomedical Telemetry: Communication between Implanted Devicesand the External World, Opticon 1826, (8): Spring, 2010).

Non-Human Animal Model of Pain

The present disclosure provides a non-human animal model of nociceptivepain, where the non-human animal expresses in a neuron (e.g., a primaryafferent neuron, such as a small- or a large-diameter primary afferentneuron; e.g., a nociceptor) of the animal a nucleic acid comprising anucleotide sequence encoding an opsin polypeptide that provides fordepolarization of the nociceptor in response to light of a wavelengththat activates the opsin. Illumination of the depolarizing opsin,expressed in a membrane of the neuron (e.g., primary afferent neuron,such as a small- or a large-diameter primary afferent neuron; e.g.,nociceptor) in the non-human animal, induces pain in the animal.

A subject non-human animal model is useful for identifying agents thatcontrol pain (as described below). A subject non-human animal model isuseful for research applications, e.g., to investigate the role ofnociceptor activity in the genesis of pain (e.g., neuropathic pain). Insome cases, a subject non-human animal model of pain is a rat. In somecases, a subject non-human animal model of pain is a mouse.

In some embodiments, the non-human animal model is not a transgenicanimal, e.g., the non-human animal model does not include a nucleic acidencoding a light-activated polypeptide integrated into the genome of agerm cell. In some embodiments, the non-human animal model comprises anucleic acid encoding a light-activated polypeptide in a primaryafferent neuron, such as a nociceptor, where the nucleic acid may beintegrated into the genome of the primary afferent neuron (e.g.,nociceptor). In some embodiments, the non-human animal model comprises anucleic acid encoding a light-activated polypeptide in a primaryafferent neuron (e.g., nociceptor), and not in a non-neuronal cell,where the nucleic acid is integrated into the genome of the nociceptor.In some embodiments, the non-human animal model comprises a nucleic acidencoding a light-activated polypeptide in a primary afferent neuron(e.g., nociceptor), and not in a non-neuronal cell of the animal, wherethe nucleic acid is not integrated into the genome of the nociceptor.

In some cases, a Cre-dependent DIO-AAV6 construct (see, e.g., Sohal etal. (2009) Nature 459:698) comprising a nucleotide sequence encoding adepolarizing light-activated polypeptide can be used with anociceptor-specific Cre mouse line, to achieve opsin expressionrestricted to sub-populations of nociceptors. For example, a nucleotidesequence encoding a depolarizing light-responsive polypeptide isincluded within a Cre-dependent DIO-AAV6 construct; and the construct isintroduced into nociceptors in a nociceptor-specific Cre mouse.

Depolarizing Light-Activated Proteins

As discussed above, a subject non-human animal model of pain expressesin a neuron (e.g., a primary afferent neuron, such as a small- or alarge-diameter primary afferent neuron; e.g., a nociceptor) of theanimal a nucleic acid comprising a nucleotide sequence encoding an opsinpolypeptide that provides for depolarization of the nociceptor inresponse to light of a wavelength that activates the opsin.

Examples of suitable light-responsive polypeptides include, e.g.,members of the Channelrhodopsin family of light-responsive cationchannel proteins such as Chlamydomonas rheinhardtii channelrhodopsin 2(ChR2); a step-function opsin (SFO); a stabilized SFO (SSFO); a chimericopsin such as C1V1; a Volvox carteri-derived channelrhodopsin (VChR1),etc. Such light-responsive polypeptides can be used to promote neuralcell membrane depolarization in response to a light stimulus.

Enhanced Intracellular Transport Amino Acid Motifs

Light-responsive opsin proteins having components derived fromevolutionarily simpler organisms may not be expressed or tolerated bymammalian cells or may exhibit impaired subcellular localization whenexpressed at high levels in mammalian cells. Consequently, in someembodiments, the light-responsive opsin proteins expressed in a cell canbe fused to one or more amino acid sequence motifs selected from thegroup consisting of a signal peptide, an endoplasmic reticulum (ER)export signal, a membrane trafficking signal, and/or an N-terminal golgiexport signal. The one or more amino acid sequence motifs which enhancelight-responsive protein transport to the plasma membranes of mammaliancells can be fused: a) to the N-terminus of the light-responsiveprotein; b) to the C-terminus of the light-responsive protein; c) toboth the N- and C-terminal ends of the light-responsive protein; or d)internally within the light-responsive protein. Optionally, thelight-responsive protein and the one or more amino acid sequence motifsmay be separated by a linker.

In some embodiments, the light-responsive protein can be modified by theaddition of a trafficking signal (ts) which enhances transport of theprotein to the cell plasma membrane. In some embodiments, thetrafficking signal can be derived from the amino acid sequence of thehuman inward rectifier potassium channel Kir2.1. In other embodiments,the trafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Trafficking sequences that aresuitable for use can comprise an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence such a traffickingsequence of human inward rectifier potassium channel Kir2.1 (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)).

A trafficking sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Signal sequences that are suitable for inclusion in a light-activatedpolypeptide can comprise an amino acid sequence having at least 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acidsequence identity to an amino acid sequence such as one of thefollowing:

1) the signal peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ IDNO:23))

2) the P2 subunit signal peptide of the neuronal nicotinic acetylcholinereceptor (e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:24));

3) a nicotinic acetylcholine receptor signal sequence (e.g.,MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:25)); and

4) a nicotinic acetylcholine receptor signal sequence (e.g.,MRGTPLLLVVSLFSLLQD (SEQ ID NO:26)).

A signal sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Endoplasmic reticulum (ER) export sequences that are suitable for use ina light-responsive polypeptide include, e.g., VXXSL (where X is anyamino acid) (e.g., VKESL (SEQ ID NO:27); VLGSL (SEQ ID NO:28); etc.);NANSFCYENEVALTSK (SEQ ID NO:29); FXYENE (SEQ ID NO:30) (where X is anyamino acid), e.g., FCYENEV (SEQ ID NO:31); and the like. An ER exportsequence can have a length of from about 5 amino acids to about 25 aminoacids, e.g., from about 5 amino acids to about 10 amino acids, fromabout 10 amino acids to about 15 amino acids, from about 15 amino acidsto about 20 amino acids, or from about 20 amino acids to about 25 aminoacids.

In some embodiments, the native signal peptide sequence in the proteincan be deleted or substituted with a heterologous signal peptidesequence from a different protein.

ChR

In some aspects, the light-responsive cation channel protein can bederived from Chlamydomonas reinhardtii, wherein the cation channelprotein can be capable of transporting cations across a cell membranewhen the cell is illuminated with light. In another embodiment, thelight-responsive cation channel protein can comprise an amino acidsequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:8. The lightused to activate the light-responsive cation channel protein derivedfrom Chlamydomonas reinhardtii can have a wavelength between about 460and about 495 nm or can have a wavelength of about 480 nm. Additionally,light pulses having a temporal frequency of about 100 Hz can be used toactivate the light-responsive protein. In some embodiments, activationof the light-responsive cation channel derived from Chlamydomonasreinhardtii with light pulses having a temporal frequency of about 100Hz can cause depolarization of the neurons expressing thelight-responsive cation channel. The light-responsive cation channelprotein can additionally comprise substitutions, deletions, and/orinsertions introduced into a native amino acid sequence to increase ordecrease sensitivity to light, increase or decrease sensitivity toparticular wavelengths of light, and/or increase or decrease the abilityof the light-responsive cation channel protein to regulate thepolarization state of the plasma membrane of the cell. Additionally, thelight-responsive cation channel protein can contain one or moreconservative amino acid substitutions and/or one or morenon-conservative amino acid substitutions. The light-responsive protonpump protein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to transport cations across a cell membrane.

In some embodiments, the light-responsive cation channel comprises aT159C substitution of the amino acid sequence set forth in SEQ ID NO:8.In some embodiments, the light-responsive cation channel comprises aL132C substitution of the amino acid sequence set forth in SEQ ID NO:8.In some embodiments, the light-responsive cation channel comprises anE123T substitution of the amino acid sequence set forth in SEQ ID NO:8.In some embodiments, the light-responsive cation channel comprises anE123A substitution of the amino acid sequence set forth in SEQ ID NO:8.In some embodiments, the light-responsive cation channel comprises aT159C substitution and an E123T substitution of the amino acid sequenceset forth in SEQ ID NO:8. In some embodiments, the light-responsivecation channel comprises a T159C substitution and an E123A substitutionof the amino acid sequence set forth in SEQ ID NO:8. In someembodiments, the light-responsive cation channel comprises a T159Csubstitution, an L132C substitution, and an E123T substitution of theamino acid sequence set forth in SEQ ID NO:8. In some embodiments, thelight-responsive cation channel comprises a T159C substitution, an L132Csubstitution, and an E123A substitution of the amino acid sequence setforth in SEQ ID NO:8. In some embodiments, the light-responsive cationchannel comprises an L132C substitution and an E123T substitution of theamino acid sequence set forth in SEQ ID NO:8. In some embodiments, thelight-responsive cation channel comprises an L132C substitution and anE123A substitution of the amino acid sequence set forth in SEQ ID NO:8.

In some embodiments, a ChR2 protein comprises at least one (such as one,two, three, or more) amino acid sequence motifs that enhance transportto the plasma membranes of target cells selected from the groupconsisting of a signal peptide, an ER export signal, and a membranetrafficking signal. In some embodiments, the ChR2 protein comprises anN-terminal signal peptide and a C-terminal ER export signal. In someembodiments, the ChR2 protein comprises an N-terminal signal peptide anda C-terminal trafficking signal. In some embodiments, the ChR2 proteincomprises an N-terminal signal peptide, a C-terminal ER export signal,and a C-terminal trafficking signal. In some embodiments, the ChR2protein comprises a C-terminal ER export signal and a C-terminaltrafficking signal. In some embodiments, the C-terminal ER export signaland the C-terminal trafficking signal are linked by a linker. The linkercan comprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linkermay further comprise a fluorescent protein, for example, but not limitedto, a yellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

In some embodiments, the trafficking signal can be derived from theamino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Traffickingsequences that are suitable for use can comprise an amino acid sequencehaving at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)).

In some cases, the ER export signal is, e.g., VXXSL (where X is anyamino acid) (e.g., VKESL (SEQ ID NO:27); VLGSL (SEQ ID NO:28); etc.);NANSFCYENEVALTSK (SEQ ID NO:29); FXYENE (SEQ ID NO:30) (where X is anyamino acid), e.g., FCYENEV (SEQ ID NO:31); and the like.

Step Function Opsins and Stabilized Step Function Opsins Based on ChR2

In other embodiments, the light-responsive polypeptide is a stepfunction opsin (SFO) protein or a stabilized step function opsin (SSFO)protein that can have specific amino acid substitutions at key positionsin the retinal binding pocket of the protein. In some embodiments, theSFO protein can have a mutation at amino acid residue C128 of SEQ IDNO:8. In other embodiments, the SFO protein has a CI28A mutation in SEQID NO:8. In other embodiments, the SFO protein has a C128S mutation inSEQ ID NO:8. In another embodiment, the SFO protein has a C128T mutationin SEQ ID NO:8. In some embodiments, the SFO protein can comprise anamino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO:9, and comprises an alanine, senile, or threonine at amino acid 128.

In some embodiments, the SSFO protein can have a mutation at amino acidresidue D156 of SEQ ID NO:8. In other embodiments, the SSFO protein canhave a mutation at both amino acid residues C128 and D156 of SEQ IDNO:8. In one embodiment, the SSFO protein has an C128S and a D156Amutation in SEQ ID NO:8. In another embodiment, the SSFO protein cancomprise an amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO:10; and comprises an alanine, serine, or threonine at aminoacid 128; and comprises a alanine at amino acid 156. In anotherembodiment, the SSFO protein can comprise a C128T mutation in SEQ IDNO:8. In some embodiments, the SSFO protein comprises C128T and D156Amutations in SEQ ID NO:8.

In some embodiments the SFO or SSFO proteins provided herein can becapable of mediating a depolarizing current in the cell when the cell isilluminated with blue light. In other embodiments, the light can have awavelength of about 445 nm. Additionally, in some embodiments the lightcan be delivered as a single pulse of light or as spaced pulses of lightdue to the prolonged stability of SFO and SSFO photocurrents. In someembodiments, activation of the SFO or SSFO protein with single pulses orspaced pulses of light can cause depolarization of a neuron expressingthe SFO or SSFO protein. In some embodiments, each of the disclosed stepfunction opsin and stabilized step function opsin proteins can havespecific properties and characteristics for use in depolarizing themembrane of a neuronal cell in response to light.

Further disclosure related to SFO or SSFO proteins can be found inInternational Patent Application Publication No. WO 2010/056970, thedisclosure of which is hereby incorporated by reference in its entirety.

In some cases, the ChR2-based SFO or SSFO comprises a membranetrafficking signal and/or an ER export signal. In some embodiments, thetrafficking signal can be derived from the amino acid sequence of thehuman inward rectifier potassium channel Kir2.1. In other embodiments,the trafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Trafficking sequences that aresuitable for use can comprise an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence such a traffickingsequence of human inward rectifier potassium channel Kir2.1 (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ER exportsignal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ ID NO:29);FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:31); and the like.

C1V1 Chimeric Cation Channels

In other embodiments, the light-responsive cation channel protein can bea C1V1 chimeric protein derived from the VChR1 protein of Volvox carteriand the ChR1 protein from Chlamydomonas reinhardti, wherein the proteincomprises the amino acid sequence of VChR1 having at least the first andsecond transmembrane helices replaced by the first and secondtransmembrane helices of ChR1; is responsive to light; and is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments, the C1V1 protein canfurther comprise a replacement within the intracellular loop domainlocated between the second and third transmembrane helices of thechimeric light responsive protein, wherein at least a portion of theintracellular loop domain is replaced by the corresponding portion fromChR1. In another embodiment, the portion of the intracellular loopdomain of the C1V1 chimeric protein can be replaced with thecorresponding portion from ChR1 extending to amino acid residue A145 ofthe ChR1. In other embodiments, the C1V1 chimeric protein can furthercomprise a replacement within the third transmembrane helix of thechimeric light responsive protein, wherein at least a portion of thethird transmembrane helix is replaced by the corresponding sequence ofChR1. In yet another embodiment, the portion of the intracellular loopdomain of the C1V1 chimeric protein can be replaced with thecorresponding portion from ChR1 extending to amino acid residue W163 ofthe ChR1. In other embodiments, the C1V1 chimeric protein can comprisean amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO:11.

In some embodiments, the C1V1 protein can mediate a depolarizing currentin the cell when the cell is illuminated with green light. In otherembodiments, the light can have a wavelength of between about 540 nm toabout 560 nm. In some embodiments, the light can have a wavelength ofabout 542 nm. In some embodiments, the C1V1 chimeric protein is notcapable of mediating a depolarizing current in the cell when the cell isilluminated with violet light. In some embodiments, the chimeric proteinis not capable of mediating a depolarizing current in the cell when thecell is illuminated with light having a wavelength of about 405 nm.Additionally, in some embodiments, light pulses having a temporalfrequency of about 100 Hz can be used to activate the C1V1 protein.

In some cases, the C1V1 polypeptide comprises a membrane traffickingsignal and/or an ER export signal. In some embodiments, the traffickingsignal can be derived from the amino acid sequence of the human inwardrectifier potassium channel Kir2.1. In other embodiments, thetrafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Trafficking sequences that aresuitable for use can comprise an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence such a traffickingsequence of human inward rectifier potassium channel Kir2.1 (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ER exportsignal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ ID NO:29);FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:31); and the like.

C1V1 Variants

In some aspects, a suitable light-responsive polypeptide comprisessubstituted or mutated amino acid sequences, wherein the mutantpolypeptide retains the characteristic light-activatable nature of theprecursor C1V1 chimeric polypeptide but may also possess alteredproperties in some specific aspects. For example, the mutantlight-responsive C1V1 chimeric proteins described herein can exhibit anincreased level of expression both within an animal cell or on theanimal cell plasma membrane; an altered responsiveness when exposed todifferent wavelengths of light, particularly red light; and/or acombination of traits whereby the chimeric C1V1 polypeptide possess theproperties of low desensitization, fast deactivation, low violet-lightactivation for minimal cross-activation with other light-responsivecation channels, and/or strong expression in animal cells.

Accordingly, provided herein are C1V1 chimeric light-responsive opsinproteins that can have specific amino acid substitutions at keypositions throughout the retinal binding pocket of the VChR1 portion ofthe chimeric polypeptide. In some embodiments, the C1V1 protein can havean amino acid substitution at amino acid residue E122 of SEQ ID NO:11.In some embodiments, the C1V1 protein can have a substitution at aminoacid residue E162 of SEQ ID NO:11. In other embodiments, the C1V1protein can have a substitution at both amino acid residues E162 andE122 of SEQ ID NO:11. In other embodiments, the C1V1 protein cancomprise an amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; and can include one or moreof the aforementioned amino acid substitutions.

In some aspects, the C1V1-E122 mutant chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In other embodiments, the C1V1-E122 mutantchimeric protein can mediate a depolarizing current in the cell when thecell is illuminated with red light. In some embodiments, the red lightcan have a wavelength of about 630 nm. In some embodiments, theC1V1-E122 mutant chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with violet light. Insome embodiments, the chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with light having awavelength of about 405 nm. Additionally, in some embodiments, lightpulses having a temporal frequency of about 100 Hz can be used toactivate the C1V1-E122 mutant chimeric protein. In some embodiments,activation of the C1V1-E122 mutant chimeric protein with light pulseshaving a frequency of 100 Hz can cause depolarization of the neuronsexpressing the C1V1-E122 mutant chimeric protein.

In other aspects, the C1V1-E162 mutant chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 535 nm to about 540 nm. In some embodiments, the light can have awavelength of about 542 nm. In other embodiments, the light can have awavelength of about 530 nm. In some embodiments, the C1V1-E162 mutantchimeric protein does not mediate a depolarizing current in the cellwhen the cell is illuminated with violet light. In some embodiments, thechimeric protein does not mediate a depolarizing current in the cellwhen the cell is illuminated with light having a wavelength of about 405nm. Additionally, in some embodiments, light pulses having a temporalfrequency of about 100 Hz can be used to activate the C1V1-E162 mutantchimeric protein. In some embodiments, activation of the C1V1-E162mutant chimeric protein with light pulses having a frequency of 100 Hzcan cause depolarization-induced synaptic depletion of the neuronsexpressing the C1V1-E162 mutant chimeric protein.

In yet other aspects, the C1V1-E122/E162 mutant chimeric protein iscapable of mediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In some embodiments, the C1V1-E122/E162mutant chimeric protein does not mediate a depolarizing current in thecell when the cell is illuminated with violet light. In someembodiments, the chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with light having awavelength of about 405 nm. In some embodiments, the C1V1-E122/E162mutant chimeric protein can exhibit less activation when exposed toviolet light relative to C1V1 chimeric proteins lacking mutations atE122/E162 or relative to other light-responsive cation channel proteins.Additionally, in some embodiments, light pulses having a temporalfrequency of about 100 Hz can be used to activate the C1V1-E122/E162mutant chimeric protein. In some embodiments, activation of theC1V1-E122/E162 mutant chimeric protein with light pulses having afrequency of 100 Hz can cause depolarization-induced synaptic depletionof the neurons expressing the C1V1-E122/E162 mutant chimeric protein.

In some cases, the C1V1 variant polypeptide comprises a membranetrafficking signal and/or an ER export signal. In some embodiments, thetrafficking signal can be derived from the amino acid sequence of thehuman inward rectifier potassium channel Kir2.1. In other embodiments,the trafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Trafficking sequences that aresuitable for use can comprise an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence such a traffickingsequence of human inward rectifier potassium channel Kir2.1 (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ER exportsignal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ ID NO:29);FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:31); and the like.

Volvox Carteri Light-Activated Polypeptide

In some embodiments, a suitable light-responsive polypeptide is a cationchannel derived from Volvox carteri (VChR1) and is activated byillumination with light of a wavelength of from about 500 nm to about600 nm, e.g., from about 525 nm to about 550 nm, e.g., 545 nm. In someembodiments, the light-responsive ion channel protein can comprise anamino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO:19. The light-responsive ion channel protein can additionallycomprise substitutions, deletions, and/or insertions introduced into anative amino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive ionchannel protein to regulate the polarization state of the plasmamembrane of the cell. Additionally, the light-responsive ion channelprotein can contain one or more conservative amino acid substitutionsand/or one or more non-conservative amino acid substitutions. Thelight-responsive ion channel protein comprising substitutions,deletions, and/or insertions introduced into the native amino acidsequence suitably retains the ability to transport ions across theplasma membrane of a neuronal cell in response to light.

In some cases, a light-responsive cation channel protein can comprise acore amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQID NO:19 and at least one (such as one, two, three, or more) amino acidsequence motifs which enhance transport to the plasma membranes ofmammalian cells selected from the group consisting of a signal peptide,an ER export signal, and a membrane trafficking signal. In someembodiments, the light-responsive proton ion channel comprises anN-terminal signal peptide and a C-terminal ER export signal. In someembodiments, the light-responsive ion channel protein comprises anN-terminal signal peptide and a C-terminal trafficking signal. In someembodiments, the light-responsive ion channel protein comprises anN-terminal signal peptide, a C-terminal ER Export signal, and aC-terminal trafficking signal. In some embodiments, the light-responsiveion channel protein comprises a C-terminal ER Export signal and aC-terminal trafficking signal. In some embodiments, the C-terminal ERExport signal and the C-terminal trafficking signal are linked by alinker. The linker can comprise any of about 5, 10, 20, 30, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker may further comprise a fluorescent protein, forexample, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments the ER Export signal is more C-terminallylocated than the trafficking signal. In some embodiments the traffickingsignal is more C-terminally located than the ER Export signal.

In some embodiments, the trafficking signal can be derived from theamino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Traffickingsequences that are suitable for use can comprise an amino acid sequencehaving at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ERexport signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL(SEQ ID NO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ IDNO:29); FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV(SEQ ID NO:31); and the like.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive channel proteins described herein, such as alight-responsive ion channel protein comprising a core amino acidsequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the sequence shown in SEQ ID NO:19. Also providedherein are expression vectors (such as a viral vector described herein)comprising a polynucleotide encoding the proteins described herein, suchas a light-responsive channel protein comprising a core amino acidsequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the sequence shown in SEQ ID NO:19.

Step Function Opsins and Stabilized Step Function Opsins Based on VChR1

In other embodiments, the light-responsive polypeptide is a SFO or anSSFO based on VChR1.

In some embodiments, the SFO protein can have a mutation at amino acidresidue C123 of SEQ ID NO:19. In other embodiments, the SFO protein hasa C123A mutation in SEQ ID NO:19. In other embodiments, the SFO proteinhas a C123S mutation in SEQ ID NO:19. In another embodiment, the SFOprotein has a C123T mutation in SEQ ID NO:19. In some embodiments, theSFO protein can comprise an amino acid sequence at least 75%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:20, and comprises an alanine, serine, orthreonine at amino acid 123.

In some embodiments, the SFO protein can have a mutation at amino acidresidue D151 of SEQ ID NO:19. In other embodiments, the SFO protein canhave a mutation at both amino acid residues C123 and D151 of SEQ IDNO:19. In one embodiment, the SFO protein has an C123S and a D151Amutation in SEQ ID NO:19. In another embodiment, the SSFO protein cancomprise an amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO:21; and comprises an alanine, serine, or threonine at aminoacid 122; and comprises a alanine at amino acid 151.

In some embodiments the SFO or SSFO proteins provided herein can becapable of mediating a depolarizing current in the cell when the cell isilluminated with blue light. In other embodiments, the light can have awavelength of about 560 nm. Additionally, in some embodiments the lightcan be delivered as a single pulse of light or as spaced pulses of lightdue to the prolonged stability of SFO and SSFO photocurrents. In someembodiments, activation of the SFO or SSFO protein with single pulses orspaced pulses of light can cause depolarization of a neuron expressingthe SFO or SSFO protein. In some embodiments, each of the disclosed stepfunction opsin and stabilized step function opsin proteins can havespecific properties and characteristics for use in depolarizing themembrane of a neuronal cell in response to light.

In some cases, the VChR1-based SFO or SSFO comprises a membranetrafficking signal and/or an ER export signal. In some embodiments, thetrafficking signal can be derived from the amino acid sequence of thehuman inward rectifier potassium channel Kir2.1. In other embodiments,the trafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:22). Trafficking sequences that aresuitable for use can comprise an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence such a traffickingsequence of human inward rectifier potassium channel Kir2.1 (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:22)). In some cases, the ER exportsignal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:27); VLGSL (SEQ ID NO:28); etc.); NANSFCYENEVALTSK (SEQ ID NO:29);FXYENE (SEQ ID NO:30) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:31); and the like.

Screening Methods

The present disclosure provides methods of identifying an agent that issuitable for use in controlling pain.

Methods of Identifying an Agent that Reduces Pain

In some cases, a subject method involves: a) contacting a non-humananimal (e.g., a non-human mammal such as a rat or a mouse) of thepresent disclosure with a test agent, where the non-human animalexpresses a depolarizing light-activated polypeptide in a primaryafferent neuron, such as a nociceptor; and b) determining the effect, ifany, of the test agent on pain when the depolarizing light-activatedpolypeptide is illuminated (activated) with light. A test agent thatreduces pain in the non-human animal, compared to the level of paininduced by light activation of the depolarizing light-activatedpolypeptide in the absence of the test agent, indicates that the testagent is a candidate agent for controlling (reducing) pain. In somecases, the non-human animal is a subject non-human animal model, asdescribed above.

For example, a test agent that reduces pain by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, or more than 25% (e.g., 25% to 50%; 50% to 75%; etc.) is considereda candidate agent for reducing pain.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The terms “candidate agent,” “test agent,” “agent”, “substance” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.) and can also be used. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available from Pan Labs (Bothell, Wash.) or are readily producible.

Candidate agents can be small organic or inorganic compounds having amolecular weight of more than 50 daltons and less than about 2,500daltons. Candidate agents can comprise functional groups necessary forstructural interaction with proteins, e.g., hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, and derivatives, structural analogs orcombinations thereof.

Assays of the present disclosure include controls, where suitablecontrols include a non-human animal model that expresses a depolarizinglight-responsive polypeptide in a nociceptor, and that has been exposedto activating light, but has not been administered the test agent.

Whether a test agent reduces pain in the non-human animal can bedetermined using any of a variety of assays known in the art. Forexample, test that measure pain through one or more behaviors such aswithdrawal, licking, immobility, and vocalization, can be used. Suitabletests include, e.g.: a) the formalin assay; b) the von Frey test; c) athermal assay such as tail withdrawal assay, a hot plate assay, a tailflick test (Rao et al. (1996) Neuropharmacol. 35:393); d) the Hargreavesassay; and the like. See, e.g., Mogil, et al. (2001) Methods in PainResearch, Frontiers in Neuroscience; and Carter and Shieh (2010)Nociception: Guide to Research Techniques in Neuroscience, Burlington,Mass., Academic Press, pp 51-52; and Bannon and Malmberg (2007) CurrentProtocols in Neuroscience, Wiley Online Library. Suitable tests includethose described in the Examples.

Methods of Identifying Agents that Increase the Minimum Intensity ofLight Required to Produce a Pain Response

The present disclosure provides a method of identifying an agent thatreduces pain, the method comprising: a) contacting a non-human animal(e.g., a non-human mammal such as a rat or a mouse) of the presentdisclosure with a test agent, where the non-human animal expresses adepolarizing light-activated polypeptide in a primary afferent neuron,such as a nociceptor; and b) determining the effect, if any, of the testagent on the minimum amount of light required to induce pain followingadministration of the test agent.

A test agent that increases the amount of light required to induce painis a candidate agent for reducing pain. For example, a test agent thatincreases the amount of light (as expressed in mW/mm²) required toinduce pain by at least 10%, at least 15%, at least 20%, at least 25%,at least 50%, at least 75%, at least 100% (or 2-fold), at least2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold, isconsidered a candidate agent for reducing pain.

For example, if the minimum light intensity of light required to producea pain response in the non-human animal is 0.5 mW/mm², and the testagent increased the minimum amount of light required to induce pain to0.75 mW/mm², the test agent would be considered a candidate agent forreducing pain.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The terms “candidate agent,” “test agent,” “agent”, “substance” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.) and can also be used. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available from Pan Labs (Bothell, Wash.) or are readily producible.

Candidate agents can be small organic or inorganic compounds having amolecular weight of more than 50 daltons and less than about 2,500daltons. Candidate agents can comprise functional groups necessary forstructural interaction with proteins, e.g., hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, and derivatives, structural analogs orcombinations thereof.

Assays of the present disclosure include controls, where suitablecontrols include a non-human animal model that expresses a depolarizinglight-responsive polypeptide in a nociceptor, and that has been exposedto activating light, but has not been administered the test agent. Insome cases, a control is a non-human animal model that has beenadministered an agent known not to affect pain.

Whether a test agent increases the minimum amount of light required toinduce a pain response in the non-human animal can be determined usingany of a variety of assays known in the art. For example, test thatmeasure pain through one or more behaviors such as withdrawal, licking,immobility, and vocalization, can be used. Suitable tests include, e.g.:a) the formalin assay; b) the von Frey test; c) a thermal assay such astail withdrawal assay, a hot plate assay, a tail flick test (Rao et al.(1996) Neuropharmacol. 35:393); d) the Hargreaves assay; and the like.See, e.g., Mogil, et al. (2001) Methods in Pain Research, Frontiers inNeuroscience; and Carter and Shieh (2010) Nociception: Guide to ResearchTechniques in Neuroscience, Burlington, Mass., Academic Press, pp 51-52;and Bannon and Malmberg (2007) Current Protocols in Neuroscience, WileyOnline Library. Suitable tests include those described in the Examples.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Bidirectional Control of Pain

It is demonstrated here that optogenetics can be used to bidirectionallycontrol acute pain in both normal and pathological states. A genetransduction strategy was used that is adaptable and clinicallyrelevant. Adeno-associated virus serotype 6 (AAV6) was used. AAV6 hasmany attractive features; it has been used for gene delivery innon-human primates, and is a leading candidate for future use in humanclinical trials¹⁵. It is also capable of retrograde transport, and canspecifically transduce nociceptors through intraneural delivery,removing the need for risky dorsal root ganglion injections.

Materials and Methods Animal Test Subjects and Experiments

All surgical and behavioral procedures were approved by the StanfordUniversity Administrative Panel on Lab Animal Care. Female C57BL/6 mice(1-4 months old) were housed in groups of 5 under a 12:12 light:darkcycle. Food and water were available ad libitum.

Intraneural Injection of AAV6-hSyn-ChR2(H134R)-eYFP andAAV6-hSyn-eNpHR3.0-eYFP

Mice were anesthetized with 2-2.5% isoflurane, placed on a heating padmaintained at 37° C., and allowed to reach a stable plane of anesthesia,which was periodically checked through examination of breathing rate anda toe-pinch test. Fur was shaved from the femur, bilaterally orunilaterally, depending on the injection, using an electrical razor. Ahair removal cream (Nair) was used to remove any remaining hair from theincision site. The incision site was then sterilized with alternatingapplications of ethanol and Betadine solution, and the mouse legs tapedto the surgical table. 100 μl of 1 mg/ml Rimadyl was injected.Sterilized forceps and spring scissors were then used to make a 2 cmincision immediately above the femur. The gluteus superficialis andbiceps femoris muscles were identified and the connective tissue betweenthem cut to expose the sciatic nerve cavity. Retractors were used tokeep the cavity open and allow for clear access to the nerve. The nervewas carefully freed from the underlying fascia using bluntedmicromanipulators and spring scissors. 100 μl of 0.25% Bupivacaine wasinjected into the incision site to simultaneously prevent the nerve fromdrying and induce local anesthesia. A 35G beveled needle (Nanofil #NF35BV-2, World Precision Instruments) was carefully inserted into thenerve, and 2.5-4 μl of virus solution injected at 1 μl/min, using a 25μl syringe (Hamilton Company), connected to a Harvard PHD Syringe pump(Harvard Apparatus). When possible, 2 separate injections were made intothe common peroneal and tibial branches of the sciatic nerve, to ensurethat the nerve was filled uniformly. ChR2 injected mice received 3×10¹⁰vg (from UNC Vector Core), while NpHR injected mice received either1×10¹¹ vg or 3×10¹¹ vg (from UNC Vector Core and Virovek, respectively).Depending on the mouse, this procedure was performed either unilaterallyor bilaterally. The incision was then sutured closed using 5-0 suture.

Isolation, Culture and Electrophysiology of Opsin-Expressing DRG NeuronsIsolation of DRG

Dorsal root ganglion (DRG) excision, culture and electrophysiologyprocedures were largely based on previously reported protocols²³. Mice,three to four weeks after intraneural injection, were deeplyanesthetized with isoflurane 5% and fur was shaved from the back. Micewere then perfused with 4° C. sterile phosphate buffered saline. Thefollowing isolation steps were rapidly performed, and completed within 5minutes after perfusion. After removing the skin from the back, usingsterile procedure, the muscles along the vertebral column were cut andbone rongeurs used to peel away any muscle or tendon superficial to thevertebrae. The rongeurs were used to break away the vertebral bonedirectly dorsal to the spinal cord, starting at the base of the spine,and moving rostrally. Muscle lateral to the spinal cord was peeled awayuntil the sciatic nerve branches could be visualized, and bones werebroken lateral to the spinal cord to free the path of the nerve. Eachnerve branch was cut using small spring scissors, pulled proximally withforceps until the dorsal root ganglion could be visualized, and cutproximal to the DRG. The DRG was then placed in 4° C., sterileMEM-complete solution (minimal essential media, MEM vitamins,antibiotics, and 10% fetal bovine serum). Three DRGs were excised fromeach expressing side of the mouse.

DRG Culture

Excised DRGs were desheathed and transferred to MEM-Collagenase solution(minimal essential media, vitamins, antibiotics, no fetal bovine serum,0.125% collagenase). The tissue was incubated at 37° C. for 45 minutesin a water bath and then triturated in 2.5 ml TripleE Express(Invitrogen). The trypsin was quenched with 2.5 ml MEM-complete with 80ug/ml DNase I, 100 μg/ml trypsin inhibitor from chicken egg white and2.5 mg/ml MgSO₄. Cells were centrifuged, and resuspended in MEM-completeat a cell density of 500,000 cells/ml. 100 ul of the cell suspension wascarefully placed as a bubble on matrigel-coated coverslips, and thenincubated at 37° C., 3% CO₂, 90% humidity. Two hours after initialincubation, the cultured neurons were flooded with 1 ml of MEM-complete.Cells were maintained 2-7 days in culture with fresh media changes asneeded until electrophysiology was performed.

Electrophysiology

A Spectra X Light engine (Lumencor) or DG4 xenon lamp (SutterInstruments) was used to identify fluorescent protein expression, anddeliver light pulses for opsin activation. A 475/28 filter was used toapply blue light for ChR2, and a 586/20 filter was used to apply yellowlight for NpHR. Light power density through the microscope objective wasmeasured with a power meter (ThorLabs). Whole-cell recordings wereobtained with patch pipettes (4-6 MO) pulled from borosilicate glasscapillaries (Sutter Instruments) with a horizontal puller (P-2000,Sutter Instruments). The external recording solution contained (in mM):125 NaCl, 2 KCl, 2 CaCl₂, 2MgCl₂, 30 glucose, 25 HEPES, and 1 μMtetrodotoxin when necessary to eliminate escape spikes for peakphotocurrent measurements. The internal recording solution contained (inmM): 130 K-gluconate, 10 mM KCl, 10 HEPES, 10 EGTA, 2 MgCl₂. Recordingswere made using a MultiClamp700B amplifier (Molecular Devices), andpClamp10.3 software (Molecular Devices) was used to record and analyzedata. Signals were filtered at 4 kHz using a Bessel filter and digitizedat 10 kHz with a Digidata 1440A analog-digital interface(MolecularDevices). Peak and steady-state photocurrents were measuredfrom a 1 s light pulse in voltage-clamp mode, where cells were held at−50 mV. Series resistances were carefully monitored and recordings werenot used if the series resistance changed significantly (by >20%) orreached 20 Ma

Latency to Light Measurement Experimental Protocol:

Approximately 1 to 5 weeks after intraneural injection, mice were placedin a plastic enclosure with a thin, transparent, plastic floor andallowed to habituate to the test setup for 30 minutes prior to testing.A multimode optical fiber (Thor Labs, # AFS105/125Y) attached to a laser(OEM Laser Systems, 473 nm, 1 mW/mm²) was directed at the footpadthrough the floor. To begin the trial, the animal was required to: (1)be awake, (2) have all four paws on the floor, and (3) be at rest, notpreparing to walk. Latency was calculated from when the footpad wasilluminated to when the paw was withdrawn. To avoid experimenter bias,no subjective criteria were applied to the end-point for latencycalculation, i.e. normal ambulation was also considered to end thetrial. A maximum latency of 1 minute was set to ensure practicality ofdata collection. Individual trials were at least 2 minutes apart, andeach mouse had five trials, which were averaged together. All trialswere video recorded at 30 frames per second and latencies calculatedthrough video analysis post-collection.

Statistics

A one-way ANOVA was used to analyze changes in ChR2+ mice's latencies inresponse to blue light in weeks 1-5 as compared with ChR2+ mice'sresponse to yellow light. Dunnett's post-hoc multiple-comparisons testwas used to determine which latencies were significantly different fromyellow-light controls. Effect sizes were calculated using g, anextension of Hedges' g for multiple groups²⁴.

Place Aversion Construction of 2 Chamber Place Aversion Setup

A 2 chamber place setup was built with an entryway connecting the two 10cm×12 cm chambers. The floor of each chamber, one red, the other blue,was illuminated with a 10×12 array of light-emitting diodes (Blue LEDs:475 nm, Red LEDs: 625 nm, Cree) and directed with mirrors such that thelight power density was equivalent in each room (0.15 mW/mm²).

Experimental Protocol

A single mouse was allowed to explore the 2 chamber set up for 10minutes prior to testing with the LED array floors turned off. Then, themouse's location was recorded using a video camera mid analyzed usingBIOBSERVE Viewer². The mouse position was recorded with the lights offfor 10 minutes, and then the lights were switched on, and the positionrecorded for a further 30 minutes.

Statistics

A two-sided paired Student's t-test was used to examine whether changesin mouse position preference between the ‘lights-off’ and ‘lights-on’condition were statistically significant. The percentage change betweenthe two conditions was then calculated for each ChR2+ and NpHR+ mice.These percentages were then compared using a two-sided, unpaired,Student's t-test for heteroscedastic populations). Effect sizes werecalculated using Hedges' g.

Measurement of Mechanical Withdrawal Thresholds

Mechanical allodynia was investigated through von Frey testing. Micewere allowed to habituate to the test setup for 1 hour prior to testing.Hairs of various forces were applied to the bottom of the foot using theUp-and-down method^(25,26) for approximately 2 seconds.

The appearance of any of the following behaviors was considered as awithdrawal response: (1) rapid flinch or withdrawal of the paw, (2)spreading of the toes, or (3) immediate licking of the paw. If theanimal moved the paw for some other reason before the end of the 2seconds, the test was considered ambiguous and repeated. Depending onthe opsin used, we then performed simultaneous illumination of themouse's paw with blue light (473 nm, 0.15 mW/mm²) or yellow light (593nm, 0.15 mW/mm²). The von Frey test was conducted by a single examinerfor all data collected, who was always blinded to whether the mice beingtested had opsin expression or not.

Statistics

Changes in von Frey threshold were tested for statistical significanceusing the non-parametric two-sided Wilcoxon signed-rank test. Effectsizes were calculated using Hedges' g.

Measurement of Thermal Withdrawal Latency

A modified Hargreaves plantar test apparatus was used to measure changesin thermal sensitivity with different types of illumination. Thestandard Hargreaves test glass plate was raised slightly to allowplacement of an LED ring above the infrared emitter. The LED ring wascalibrated to emit 0.15 mW/mm² of blue (475 nm, Cree) or yellow (590 nm,OSRAM Opto Semiconductors) light. To control for light-inducedconfounds, withdrawal latency to infrared heat when mice receivedon-spectrum illumination (blue light for ChR2 injected mice, and yellowlight for NpHR injected mice) was compared with off-spectrumillumination (vice versa). Withdrawal latency was automatically measuredbetween onset of infrared light and the first paw withdrawal. Infraredintensity was kept constant across all trials, and the tester was alwaysblinded as to whether the mice being tested had opsin expression or not.

Statistics

Changes in thermal withdrawal latency were compared between off-spectrumand on-spectrum illumination conditions using a two-sided, paired,Student's t-test. Effect sizes were calculated using Hedges' g.

Chronic Constriction Injury

The chronic constriction injury model used here was adapted from anexisting protocol²⁷. Animals were anesthetized with isoflurane and thesciatic nerve was exposed unilaterally in a similar fashion to thesciatic nerve exposure used for the intraneural injections. One 7-0prolene double-knot ligature was tied around the nerve such that theligature was just able to slide along the nerve, and the free ends ofthe suture were cut short. Non-absorbable 5-0 suture was used to closethe wound. In order to promote development of neuropathic pain, nopost-operative analgesics were administered.

Immunohistochemistry, Imaging, and Quantification of TransductionImmunohistochemistry

Mice were euthanized with 100 μl Beuthanasia-D, and transcardiallyperfused with 10 ml of 4° C. phosphate-buffered saline (1×PBS) and 10 mlof 4% paraformaldehyde (PFA). Bone rongeurs, spring scissors and forcepswere used to carefully remove the sciatic nerve, associated dorsal rootganglia and the spinal cord together from the mouse. The feet wereremoved separately. All tissue was placed in 4% PFA overnight, stored at4° C. Following this, samples were transferred to 30% sucrose (in 1×PBS)and stored for varying lengths of time (at minimum 1 day). Samples werelater dissected under microscopic guidance, and frozen separately inTissue-Tek O.C.T. Samples were cut at 20 thickness using a cryostat(Leica CM3050S), and mounted on slides. All samples were rinsed 3×10 minin 1×PBS to remove any residual OCT. For all targets except myelin,samples were then blocked in 0.3% Triton-X100, 2% Normal Donkey Serum(NDS), dissolved in 1×PBS for 1 hour. Samples were then incubatedovernight with primary antibody solutions with 0.3% Triton-X100, 5% NDS,dissolved in 1×PBS. The next day, samples were rinsed 3×10 min with1×PBS, and then incubated for 1 hour with secondary antibody solutionsdissolved in 1×PBS. Samples were then rinsed 3×10 min in 1×PBS, andcoverslipped with PVA DABCO. Primary antibodies used were Ratanti-Substance P (1:500, #556312, BD Pharmingen), Biotin-1B4 (1:50, #B-1205, Vector Laboratories), Rabbit anti-Somatostatin Receptor 2(1:250, # ab134152, Abcam) and Rabbit anti-VR1 (for TRPV1, 1:500, #ab31895, Abcam). Secondary antibodies used were Cy5 Donkey anti-Rabbit(1:500, #711-175-152, Jackson Laboratories), Cy3 Donkey anti-Rat (1:500,#711-165-152, Jackson Laboratories) and Streptavidin-Texas Red (3:100, #SA-5006, Vector Laboratories). For myelin, samples were permeabilizedfor 1 hour using 0.2% Triton-X100 dissolved in 1×PBS. Samples were thenincubated with FluoroMyelin Red (1:300, # F34652, Molecular Probes) for20 min. Following this, samples were rinsed 3×10 min in 1×PBS, andcoverslipped with PVA DABCO.

Confocal Imaging

Samples were imaged using a Leica TCS SP5 confocal scanning lasermicroscope, using a 20× oil immersion objective, and analyzed usingLeica LAS AF software. Images were later processed using Fiji^(2g),which was used to stitch together z-stacks, balance image brightness andcontrast, and modify colors to account for color-blindness.

Quantification

DRGs from 3 different mice injected with AAV6-ChR2 were examined forco-expression with Substance P, TRPV1, Somatostatin and 1B4, andexamined nerve samples from 3 different mice for co-expression withmyelin. For each marker, the percentage of marker-expressingneurons/axons that were YFP positive, and the percentage of YFP positiveneurons/axons that were positive for the given marker, were quantified.

Results

AAV6-hSyn-ChR2(H134R)-eYFP was injected into the sciatic nerve of mice(FIG. 1a ). Two to four weeks after injection, electrophysiologicalrecordings from isolated ChR2 positive neurons in the dorsal rootganglia revealed that ChR2 was functional, and expressing cells couldfire action potentials when stimulated at 5-10 Hz with 1 mW/mm² 475 nmlight (FIG. 1b ; FIG. 6). In addition, ChR2 was expressed throughout theneuron, terminating in central projections to the dorsal horn of thespinal cord, with little expression seen in deeper laminae or inascending dorsal columns, suggesting that the transduced neurons werenociceptors projecting to Rexed's lamina I/II (FIG. 1c )Immunohistochemistry showed considerable overlap between ChR2 expressionand nociceptive markers, such as 1134, Substance P, TRPV1, andSomatostatin. No overlap was observed between ChR2 and myelin,indicating that no proprioceptors were transduced in the sciatic nerve;instead, expression was restricted to unmyelinated nociceptors (putativeC-fibers) (Table I, provided in FIG. 9). Further, transduced dorsal rootganglia neurons were smaller in diameter than untransduced neurons (FIG.6), consistent with prior histological evidence regarding C-fibersize¹⁷.

The behavioral effect of optogenetic activation of these transducednociceptors was examined. Mice were allowed to freely explore a chamberwith a transparent floor. After habituation, blue light (1 mW/mm²) wasshown on the plantar hindpaws of ChR2 injected mice with the aim ofoptogenetically activating nociceptor nerve endings in the skin, andobserved characteristic pain-like behavior (FIG. 2a ). In response toblue light, mice flinched, engaged in prolonged foot-licking orvocalized: operant behaviors associated with pain¹⁸. To quantify thiseffect, the time between light onset and any paw withdrawal wasmeasured, regardless of whether such withdrawal was due to pain ornormal exploratory behavior. This latency reduced dramatically 2 weeksafter injection (P=0.034, effect size: 2.10), and remained low for 3weeks thereafter (week 3: P=0.027, effect size=2.17; week 4: P=0.026,effect size=2.19), as compared with latencies recorded from YFP-injectedmice, and ChR2 injected mice that received off-spectrum yellow lightillumination (FIG. 2b ). Latencies increased 5 weeks followinginjection, potentially due to shutdown of transgene expression in sometested mice, as has been previously reported with AAV6 in mice¹⁹.Despite stimulation being entirely transcutaneous, ChR2 injected micewere light sensitive, withdrawing in a few hundred milliseconds inresponse to low intensities of blue light (1 mW/mm²). Lower levels ofillumination showed progressively less of an effect, while an increasein illumination intensity past 1 mW/mm² did not result in any additionaldecrease in latency (FIG. 2c ).

To test if optogenetic induction of pain was tunable, it was asked iflower intensities of illumination (0.25 mW/mm²) that did not proveimmediately aversive would cause more subtle effects. A place aversionapparatus was constructed, in which the floor of each chamber wasilluminated with an LED array that emitted either off-spectrum (red, 625nm) or on-spectrum (blue, 475 nm) light (FIG. 2d ). ChR2 injected micewhen exploring the blue chamber did not show any outward signs of pain,and did not engage in foot-licking or flinch from the light, but showedan 80-20% preference for the red chamber over the blue chamber(P=0.0013, effect size=3.11). YFP injected mice showed no significantpreference (FIG. 2e,f ). Such aversion is potentially caused by lowlevels of pain that do not rise to levels that induce reflexivewithdrawal, but still cause changes in operant behavior.

It was reasoned that such low levels of optogenetic stimulation may alsoact to sensitize ChR2 injected mice to otherwise inoffensive stimuli. Todemonstrate this, von Frey testing of the mechanical withdrawalthreshold and Hargreaves testing of the thermal withdrawal latency wasconduced, but with concurrent illumination of the relevant paw with lowintensities of blue light (0.15 mW/mm², FIG. 3a, b ). While suchillumination was insufficient to induce immediate aversion (FIG. 2c ),it did significantly lower von Frey thresholds (FIG. 3b ) by 50%(P=0.027, effect size=0.904), and lower Hargreaves latency (FIG. 3f, g )by 55% (P=0.00038, effect size=2.77). Such sensitization may occurthrough sub-threshold depolarization induced in nociceptor free nerveendings, which may render them more sensitive to otherwise innocuousstimuli. Wild type mice showed no significant difference in behaviorbetween illuminated and non-illuminated tests.

To complement our ability to optogenetically induce pain, methods weredeveloped to optogenetically inhibit action potential generation innociceptors. Such inhibition could have great therapeutic value, andprovide a type of spatially and temporally restricted control overaction potential generation not possible with pharmacology or electricalstimulation.

Mice were injected in the sciatic nerve with AAV6-hSyn-eNpHR3.0-eYFP;and similar transduction profiles to our ChR2 results were observed(FIG. 7). Electrophysiological recordings of isolated cultured NpHRpositive DRG neurons revealed strong hyperpolarization in response toconstant yellow light illumination that was sufficient to block actionpotential initiation (FIG. 3c ; FIG. 8).

NpHR-injected mice were tested using similarly modified von Frey andHargreaves apparatuses that emitted yellow (593 nm) light. TheNpHR-injected mice, when illuminated with 1.1-1.7 mW/mm² light, had a69% increase (P=0.0043, effect size=0.802) in their von Frey withdrawalthresholds (FIG. 3d, e ). Low intensities of yellow light (0.15 mW/mm²)were sufficient to increase Hargreaves withdrawal latency by 97%(P=0.00019, effect size=2.05). Wild type mice showed no significantchange in behavior upon illumination with yellow light.

Finally, whether the ability to optogenetically inhibit nociception wastherapeutically relevant was determined by testing in an animal model ofneuropathic pain. Baseline von Frey and Hargreaves testing wereperformed on NpHR-injected mice, replicating our initial findings thatyellow light desensitized mice to mechanical and thermal stimuli (FIG.4a,4b ). A chronic constriction injury was performed to induce symptomsof neuropathic pain in these mice. As expected, mice showed thermal andmechanical allodynia following the injury. Mice were then illuminatedwith yellow light while performing von Frey and Hargreaves testing. Itwas observed that optogenetic inhibition could reverse mechanicalallodynia, increasing von Frey thresholds from 36 to 94% of pre-injury,non-illuminated levels (P=0.0020, effect size=0.920, FIG. 4a ).Similarly, optogenetic inhibition also reversed thermal hyperalgesia inNpHR injected mice, increasing Hargreaves withdrawal latency from 55% to128% of normal, non-illuminated levels (P=0.012, effect size=1.91, FIG.4b ). In both cases, YFP injected controls showed no significant changeswith illumination, both before, and after chronic constriction injury.

Thus, the data show that opsins can be successfully expressed with highspecificity in nociceptors through a relatively simple injectionprocedure that does not require transgenesis. Moreover, sufficientlystrong opsin expression and trafficking to achieve robust behavioraleffects through non-invasive transcutaneous illumination were observed.It is believed that this is due to expression of opsins in dermal andsubdermal free nerve endings, which can be illuminated with minimaloptical attenuation. Interestingly, effects of optogenetic illuminationon mechanical and thermal thresholds were observed even when thisillumination had low intensity. This may be due to the resting potentialand baseline excitability of free nerve endings, which may allowrelatively small optically induced membrane currents to still influencedownstream neurotransmitter release. Optogenetic effects were observedover a 3-week period, from 2 to 5 weeks following AAV6 injection.

The optogenetic capabilities reported here can be widely used byscientists who seek non-invasive ways to perturb nociceptive function.In particular, as opsin expression is specific to unmyelinatednociceptors, optogenetics could be used to understand the role ofnociceptor activity in the genesis of neuropathic pain, through chronicbidirectional optogenetic control. Researchers who seek greaterspecificity and who do not wish to develop custom transgenic mice foreach individual opsin could instead use Cre-dependent D1O-AAV6²² inconcert with any of the many different nociceptor-specific Cre lines, toachieve opsin expression restricted to sub-populations of nociceptors.Non-invasive, transcutaneous optogenetic inhibition can be used as atreatment for pain, e.g., intractable chronic pain.

FIG. 1: Intra-sciatic injection of rAAV2/6-hSyn-ChR2(H134R)-eYFPtransduced unmyelinated nociceptors that projected to spinal cord laminaI. a) Injection schematic b) Electrophysiology of dissociated ChR2+ DRGneurons. Representative whole-cell current-clamp recordings showingoptogenetically induced action potentials in response to 475 nm light (5Hz, 1 mW/mm²) and representative whole-cell voltage-clamp recordings inresponse to a light pulse (1 s, 475 nm, 1 mW/mm²). The cell is held at−50 mV. c) Expression profile at the spinal cord, DRG, nerve and foot;ChR2 is shown in green, cellular markers in magenta, overlay in white.ChR2 did not colocalize with myelinated neurons, but did colocalize withnociceptive markers including 1B4, Somatostatin, TRPV1 and Substance P.

FIG. 2: Transdermal illumination of ChR2+ mice resulted in tunablepain-like behavior. a) Experimental schematic b) Time-dependence oflight-sensitivity in ChR2+ mice (Light: 1 mw/mm²) (n=4 mice). Latenciesin response to blue light were significantly lower compared to yellowlight latencies, from week 2 to week 4 following injection (One-wayANOVA: F(6,21)=3.98; P=0.0082; Dunnett's test: P (week 2)=0.034, P (week3)=0.027, P (week 4)=0.026; effect size (week 2)=2.10, effect size (week3)=2.17, effect size (week 4)=2.19). Latencies recorded from YFP+controls and from ChR2+ mice at week 1 and week 5 were not significant(P (week 1)=0.55, P (week 5)=0.49, P (YFP+)=0.99). c) Light-sensitivitydecreased with increase in intensity of blue light, steeply untilintensity was 1 mW/mm², stabilizing past this threshold. d) Placeaversion schematic e) YFP+ mice showed a non-significant preference forthe blue-lit areas (19.9% increase in time spent in blue-lit areas,P=0.06, n=5), while ChR2 mice were significantly averse to blue-litareas (55.6% decrease in time spent in blue-lit areas, effect size=3.11,P=0.0013, n=5). Inset: these two percent changes were statisticallydifferent from each other. (P=0.00061) f) Individual traces for placeaversion data. All grouped data are shown as mean+s.e.m.

FIG. 3: Blue light sensitized ChR2 expressing mice and yellow lightdesensitized NpHR expressing mice to mechanical and thermal stimuli. a)Schematic of ChR2-mediated sensitization (0.15 mW/mm² blue lightintensity) b) von Frey thresholds reduced 50% in ChR2+ mice (effectsize=0.904, P=0.027, 11=10 paws), while wild-types showed no significantchange (P=0.50, n=10 paws). c) i) Representative whole-cellvoltage-clamp recording showing outward photocurrent in anNpHR-expressing DRG neuron, in response to a 1 s, 586 nm light pulse(indicated by yellow bar). ii) Representative whole-cell current-clamprecordings showing yellow (586 nm) light-mediated inhibition ofelectrically evoked spikes (400 pA current injection, 5 ms pulse width)in an NpHR-expressing DRG neuron. d) Schematic of NpHR-mediatedinhibition (1.1-1.7 mW/mm² light intensity) e) von Frey thresholdsincreased 69% NpHR+ mice (effect size=0.802, P=0.0043, n=24 paws) whilewild-type mice showed no significant change (P=0.71, n=20 paws). f)Schematic of optogenetic modulation of thermal thresholds. Blue/Yellowlight intensity (0.15 mW/mm²) g) Withdrawal latency to infrared stimulusdecreased 55% in ChR2+ mice during blue light illumination (effectsize=2.77, P=0.00038, n=7 paws) and increased 97% in NpHR+ mice duringyellow light illumination (effect size=2.05, P=0.00019, n=10 paws)(compared to off-spectrum illumination), while wild-type latencies didnot significantly change (P=0.91, n=9 paws, controls for ChR2+ mice,P=0.26, n=10 paws, controls for NpHR+ mice). All grouped data are shownas mean+s.e.m.

FIG. 4: Yellow light stimulation of NpHR+ mice reversed mechanicalallodynia and thermal hyperalgesia caused by a chronic constrictioninjury (CCI). a) Before CCI, yellow light significantly increased vonFrey thresholds of NpHR+ mice (78% increase, effect size=1.43, P=0.0020,n=10 paws), but not of YFP+ mice (P=0.41, n=12 paws). After CCI, vonFrey thresholds of all mice significantly reduced (NpHR+ mice, 64%reduction, effect size=1.61, P=0.0020, n=10 paws; YFP+ mice, 59%reduction, effect size=1.03, P=0.00049, n=12 paws). Yellow lightsignificantly increased von Frey thresholds of NpHR+ mice to nearpre-CCI levels (258% increase, effect size=0.92, P=0.0020, n=10 paws),but did not significantly change the thresholds of YFP+ mice (P=0.57,n=12 paws). b) Before CCI, yellow light significantly increasedwithdrawal latency to infrared stimulus in NpHR+ mice (112% increase,effect size=3.95, P=0.00025, n=7 paws, compared to off-spectrumillumination), while YFP+ mice showed no significant change (P=0.97, n=9paws). After CCI, all mice showed a reduction in withdrawal latency toinfrared stimulus during off-spectrum illumination (NpHR+ mice, 45%reduction, effect size=3.75, P=0.00077, n=7 paws; YFP+ mice, 40%reduction, effect size=2.06, P=0.0038, n=9 paws). Yellow lightsignificantly increased this latency in NpHR+ mice (132% increase,effect size=1.91, P=0.012, n=7 paws) beyond initial pre-CCI latencies,but did not significantly change latency in YFP+ mice (P=0.53, n=9paws). All grouped data are shown as mean±s.e.m.

FIG. 6: Size distribution of opsin transduction. Intra-neural injectionof a) AAV6:ChR2 and b) AAV6:NpHR results in opsin transduction that isspecific to smaller diameter nociceptors (n=205 ChR2+ neurons, 666 ChR2−neurons, n=217 NpHR+ neurons, 355 NpHR− neurons). AAV6:ChR2 transduced80% of neurons <16 μm, AAV6:NpHR transduced 75% of neurons <16 μm.

FIG. 7: Representative images of NpHR transduction observed afterintra-sciatic injection of AAV2/6-hSyn-eNpHR3.0-eYFP.

FIG. 8: Electrophysiological recording from ChR2+ and NpHR+ DRG neurons.a) Image of ChR2+ DRG neuron, scale bar 25 b) Sample whole-cellcurrent-clamp recordings showing spikes evoked by 10 Hz blue (475 nm)light pulse trains (5 ms pulse widths) in a ChR2+ DRG neuron. Cell isheld at −50 mV, light power density is 1 mW/mm². c) Summary graph ofpeak and steady-state photocurrents in response to 1 s, 475 nm bluelight. Mean±SEM is plotted, n=7. d) Image of NpHR+ DRG neuron, scale bar25 μm. e) Sample whole-cell current-clamp recordings showing yellow (586nm) light-mediated hyperpolarization in an NpHR+ DRG neuron. Cell isheld at −48 mV, light power density is 1 mW/mm². f) Summary graph ofpeak and steady-state photocurrents in response to 1 s, 586 nm yellowlight. Mean±SEM is plotted, n=10.

FIG. 9 presents Table 1. Analysis of opsin transduction profiles.Co-localization percentages calculated from analysis of DRGs from 3different ChR2+ and NphR+ mice each. Somatostatin+, VR1+ and 134+neurons are the major components of the opsin+ pool.

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Example 2: Intraneurally Injected AAV8 Selectively Transduces Neuronsthat Project to Spinal Cord Dorsal Columns and Deep Spinal Cord Laminae

Large-diameter primary afferent neurons are responsible for mediatingdiverse sensory processes including pressure, vibration, pleasurabletouch, and painful touch. In the context of pain research, these neuronsare known to be significantly modified in various chronic paindisorders. Spontaneous (‘ectopic’) firing in these neurons is thought tobe one of the major contributors to the development of inflammatory andneuropathic pain, either through their directly driving central painpathways, or through modifying spinal cord circuitry to induce centralsensitization. Optogenetic inhibition of these afferent neurons wouldreduce symptoms of neuropathic pain. Optogenetic stimulation of theseafferent neurons may also act to reduce pain in some conditions, throughmulti-step circuit processes in the spinal cord that form part of the‘pain gate’. The data shown below indicate that an adeno-associatedvirus vector (AAV8) can specifically infect large-diameter primaryafferent neurons that mediate sensation of touch.

Methods

All surgical and behavioral procedures were approved by the StanfordUniversity Administrative Panel on Lab Animal Care. Under anesthesia,the sciatic nerve of C57BL/6 mice (6-8 weeks) was exposed; and 3-5 μl ofAAV8-CMV-GFP (1-2E10 viral genomes (vg)) was injected into the exposednerve. 2-4 weeks following injection, the mice were euthanized throughtranscardial perfusion. The mice were dissected and sectioned, andtissue from the spinal cords, nerves and paws was imaged.

Results

Primary afferent neurons successfully infected by AAV8-CMV-GFP projectedto deep spinal cord laminae (FIG. 10A) in the lumbar spinal cord. Inmore rostral regions of the cord, expression was seen primarily in thefasciculus gracilis, in the dorsal columns (FIG. 10B). This expressionpersisted until the brainstem.

Example 3: Delivery of Optogenetic Proteins to Sensory Neurons of theTrigeminal Ganglion

Neuropathic pain can arise in the trigeminal ganglion and is oftencharacterized by episodic, lancinating, triggerable, often shock-likefacial pain. The disease can result from infection (e.g. herpes virus),facial trauma, stroke or surgical nerve damage. The data in this exampledemonstrate delivery of the inhibitory opsin, eNpHR3.0, to the sensoryneurons of the trigeminal ganglion in rats. The data presented in thisexample demonstrate that optogenetics can be used to control paininvolving the trigeminal ganglion.

Methods

Under anesthesia, 10-week old Sprague Dawley rats were stereotaxicallyinjected with 1×10¹¹ vg of AAV5-hSyn-eNpHR3.0 into the trigeminalganglion. Animals were euthanized 4 weeks later, and the trigeminalganglion was dissected, sectioned and imaged using a confocalmicroscope.

Results

Four weeks following direct injection of AAV5-eNpHR3.0-YFP, strongexpression of the inhibitory protein in sensory neurons of thetrigeminal ganglion was observed. These results demonstrate that primarysensory neurons other than those of spinal cord dorsal root ganglia canbe targeted with opsins and can be inhibited to control pain transmittedby these neurons. The data also demonstrate the feasibility of opsindelivery using direct injection. The data are depicted in FIG. 11.

FIG. 11. Expression of eNpHR3.0 in trigeminal ganglion sensory neuronsfollowing direct injection into ganglion. eNpHR3.0 was fused to yellowfluorescent protein to facilitate visualization (green). Neurons werestained with Nissl (red). All cell nuclei were labelled with DAPI(blue).

Example 4: Delivery of Opsins and Inhibition of Pain in Animals withPrior Neuropathic Pain

The data presented below demonstrate that AAV6 delivery of NpHR,following chronic constriction injury (CCI) and development of pain, canreduce pain.

Methods

Naïve C57Bl6 mice were habituated to the Von Frey mechanical testingprocedure and then baseline mechanical thresholds recorded. Mice werethen subjected to a CCI; 10 days following nerve injury, mechanicalthresholds were recorded and only those animals that had developedneuropathic pain (as determined by a 25% or greater reduction inmechanical thresholds) were kept in the study. AAV6 expressing eitherNpHR or YFP under control of the human synapsin promoter (5× 10¹⁰ vgtotal) was delivered to the sciatic nerve of the mice. Thirty daysfollowing NpHR or YFP delivery, mechanical thresholds were recorded inthe presence or absence of yellow light application to the targeted paw.

Results

Thirty days following AAV6 delivery we observed that application oflight to the targeted paw significantly increased mechanical thresholdlevels to pre-CC1 levels in animals expressing NpHR but not YFP. Thedata are shown in FIG. 12.

FIG. 12. Mechanical thresholds for mice expressing GFP or eNpHR3.0 innociceptive fibers using AAV6. Forty days following CCI thresholds wererecorded in the presence or absence of yellow light. Animals in theeNpHR3.0 group had significantly higher mechanical thresholds in thepresence of light (p<0.05). Mechanical thresholds in the YFP group wereunchanged.

These results demonstrate that the optogenetic approach described herecan be applied to nerves that have preexisting neuropathic pain.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method for controlling pain in an individual, the method comprisingintroducing into a primary afferent neuron of the individual a nucleicacid comprising a nucleotide sequence encoding an opsin polypeptide thatprovides for hyperpolarization of the primary afferent neuron inresponse to light of a wavelength that activates the opsin.
 2. Themethod of claim 1, wherein the primary afferent neuron is a nociceptor.3. The method of claim 1, wherein the light is delivered transdermally.4. The method of claim 1, wherein the opsin comprises an amino acidsequence having at least about 75% amino acid sequence identity to oneof SEQ ID NOs:1, 3, 4, 6, 15, and
 16. 5. The method of claim 1, whereinthe pain is neuropathic pain.
 6. The method of claim 1, wherein saidintroducing is via injection into a nerve or via intramuscularinjection, or wherein said introducing is via topical, intradermal,intravenous, intrathecal, or intrapleural administration.
 7. (canceled)8. The method of claim 2, wherein the nociceptor is one that is normallyactivated by a thermal, mechanical, or chemical stimulus.
 9. The methodof claim 1, wherein the nucleic acid is a recombinant expression vector.10.-12. (canceled)
 13. The method of claim 1, wherein the nucleotidesequence is operably linked to a promoter that provides for selectiveexpression in a neuron.
 14. (canceled)
 15. The method of claim 1,wherein the individual is a mammal.
 16. (canceled)
 17. The method ofclaim 1, wherein activation of the opsin provides for an at least 10%reduction in pain.
 18. A non-human animal model of pain, wherein thenon-human animal expresses in a primary afferent neuron of the animal anucleic acid comprising a nucleotide sequence encoding an opsinpolypeptide that provides for depolarization of the nociceptor inresponse to light of a wavelength that activates the opsin.
 19. Thenon-human animal model of claim 18, wherein the primary afferent neuronis a nociceptor.
 20. The non-human animal model of claim 18, wherein theopsin comprises an amino acid sequence having at least about 75% aminoacid sequence identity to one of SEQ ID NOs:8-14 and 19-21.
 21. Thenon-human animal model of claim 18, wherein the nucleic acid is arecombinant expression vector. 22.-24. (canceled)
 25. The non-humananimal model of claim 18, wherein the nucleotide sequence is operablylinked to a promoter that provides for selective expression in a neuron.26. The non-human animal model of claim 25, wherein the promoter is asynapsin-I promoter, a human synuclein 1 promoter, a human Thy1promoter, or a calcium/calmodulin-dependent kinase II alpha (CAMKIIα)promoter.
 27. The non-human animal model of claim 18, wherein the animalis a rat or a mouse.
 28. A method of identifying an agent that reducespain, the method comprising: a) administering a test agent to thenon-human animal of claim 18; and b) determining the effect, if any, ofthe test agent on pain when the depolarizing light-activated polypeptideis activated with light, wherein a test agent that reduces pain in thenon-human animal, compared to the level of pain induced by lightactivation of the depolarizing light-activated polypeptide in theabsence of the test agent, indicates that the test agent is a candidateagent for reducing pain.
 29. A method of identifying an agent thatreduces pain, the method comprising: a) administering a test agent tothe non-human animal of claim 18; and b) determining the effect, if any,of the test agent on the amount of light required to induce painfollowing administration of the test agent, wherein a test agent thatincreases the amount of light required to induce pain is a candidateagent for reducing pain.